PROCEEDINGS OF
INTERNATIONAL SYMPOSIUM
ON ANIMAL PRODUCTION ANDCONSERVATION FOR SUSTAINABLE
DEVELOPMENT

2018-PART 1INTERNATIONAL SYMPOSIUM ON
ANIMAL PRODUCTION AND CONSERVATIONFOR SUSTAINABLE DEVELOPMENT2018
ORGANIZER:
THE UNITED GRADUATE SCHOOL OF AGRICULTURAL SCIENCE,
GIFU UNIVERSITY-PART 2UGSAS-GU & BWEL JOINT POSTER SESSIONON AGRICULTURAL 㻌 㻌 㻌 㻌 㻌 㻌 㻌 㻌 㻌 㻌 㻌 㻌 㻌 㻌 㻌
AND BASIN WATER ENVIRONMENTAL SCIENCES
2018
CO-ORGANIZER:
GIFU UNIVERSITY REARING PROGRAM
FOR BASIN WATER ENVIRONMENTAL LEADERS

DATE: OCTOBER 17-18, 2018

VENUE: 6TH FLOOR, UGSAS BLDG. GIFU UNIVERSITY, JAPANInternational Symposium
on Animal Production and Conservation for Sustainable Development 2018
Organized by The United Graduate School of Agricultural Science, Gifu University (UGSAS-GU)PROGRAM–PART 1DAY ONE: Wednesday, October 17
Time: 9:30-19:30
Venue: Main Seminar Room (6F in UGSAS Building, Gifu University)
Time Table
9:30-10:00

Registration

10:00-10:05

Opening Remarks
Prof. Masateru Senge (Dean of UGSAS-GU)

10:05-10:10

Welcome Speech
Dr. Fumiaki Suzuki (Executive Director and Vice President of Gifu University)

10:10-10:50

Keynote Speech 01
Dr. Yoko Tsukahara (International Goat Association, Langston University)

10:50-11:30

Keynote Speech 02
Assoc. Prof. Shigeru Ninomiya (Gifu University)

11:30-11:50

Photo Shooting

11:50-13:00

Lunch BreakSession 1 ʊAnimal Production and Biotechnologyʊ
Session Chair: Prof. Masato Yayota (Gifu University)
13:00-13:25
01. Dr. Asif Mahmud (Globe Biotech Limited.)
13:25-13:50

02. Dr. Sigit Prastowo (Sebelas Maret University)

13:50-14:15

03. Dr. Shiori Miura (Niigata University㸧

14:15-14:40

04. Assoc. Prof. Mohammad Nasir Uddin (Texas A&M University)

14:40-15:20

Keynote Speech 03
Assoc. Prof. Atsushi Murai (Nagoya University)

15:20-15:30

Coffee BreakSession 2 ʊAvian Production and Reproductionʊ
Session Chair: Prof. Atsushi Iwasawa (Gifu University)
15:30-15:55
01. Assoc.Prof. Ahmed Mohamed Hanafy Hassan (Suez Canal University)
15:55-16:20

02. Dr. Hanny Cho Too(Livestock Breeding and Veterinary Department, Myanmar)

16:20-16:45

03. Prof. Anisur Rahman (University of Rajshah)17:10-19:10

Dinner Meeting (At Gifu University Restaurant (2))

DAY TWO: Thursday, October 18
Time: 9:30-17:40
Venue: Main Seminar Room (6F in UGSAS Building, Gifu University)
Time Table
9:30-10:00
10:00-10:40

Registration
Keynote Speech 04
Prof. Masato Yayota (Gifu University)

Session 3 ʊAnimal Physiology and Productionʊ
Session Chair: Assoc. Prof. Mohammad Nasir Uddin(Texas A&M University)
10:40-11:05
01. Ms. Yuli Yanti (Gifu University)
11:05-11:30

02. Dr. Adi Ratriyanto (Sebelas Maret University㸧

11:30-11:55

03. Dr. Mei Matsuzaki (Shizuoka University㸧

11:55-12:35

Keynote Speech 05
Assoc. Prof. Ikki Matsuda (Chubu University)

12:15-13:45

Lunch BreakSession 4 ʊAnimal Conservationʊ
13:45-14:10

Session Chair: Prof. Shuichi Matsumura (Gifu University)
01. Dr. Rizaldi (Andalas University)

14:10-14:35

02. Prof. Irmanida Batubara (Bogor Agricultural University)

14:35-15:00

03. Dr. Dewi Imelda Roesma (Andalas University)

15:00-15:40

Break & Preparation for Poster Presentation Session

15:40-17:40-PART 2*Please refer to the next page for details.
UGSAS-GU & BWEL Joint Poster Session on Agricultural and Basin Water
Environmental Sciences 2018

UGSAS-GU & BWEL Joint Poster Session on Agricultural andBasin Water Environmental Sciences 2018
Organized by The United Graduate School of Agricultural Science, Gifu University (UGSAS-GU)
Gifu University Rearing Program for Basin Water Environmental Leaders (BWEL)PROGRAM–PART 2DAY TWO: Thursday, October 18
Time: 15:40-17:40
Venue: Main Seminar Room (6F in UGSAS Building, Gifu University)
Time Table
15:40-16:45

Poster Presentation

17:20-17:35

Best Presentation Award Ceremony

17:30-17:40

Closing Remarks
Prof. Fusheng Li (Head of the Promotion Office of Gifu University Rearing
Program for Basin Water Environmental Leaders)Presenters
P01: Siyu Chen (UGSAS-GU; BWEL)
P02: Masaya Toyoda (Graduate School of Engineering, Gifu University; BWEL)
P03: Cahyo Wisnu Rubiyanto (UGSAS-GU; BWEL)
P04: Wenjiao Li (Graduate School of Engineering, Gifu University; BWEL)
P05: Wenzhuo Feng (UGSAS-GU; BWEL)
P06: Ruoming Cao (UGSAS-GU; BWEL)
P07: Huijuan Shao (UGSAS-GU; BWEL)
P08: Ali Rahmat (UGSAS-GU; BWEL)
P09: Shuailei Li (Graduate School of Natural Science and Technology, Gifu University; BWEL)
P10: Yuto Ito (Graduate School of Natural Science and Technology, Gifu University; BWEL)
P11: Fenglan Wang (UGSAS-GU; BWEL)
P12: Souzhu (UGSAS-GU; BWEL)
P13: Jobaida Akther (UGSAS-GU; University of Dhaka)
P14: Sharmin Sultana (UGSAS-GU)
P15: Tran Duy Quan (UGSAS-GU)
P16: Raj Kishan Agrahari (UGSAS-GU)
P17: Hend Altaib (UGSAS-GU)
P18: Panyapon Pumkaeo (UGSAS-GU)
P19: Muhammad Arifin (Graduate School of Natural Science and Technology, Gifu University)
P20: Yolani Syaputri (UGSAS-GU)
P21: Nayla Majeda Alfarafisa (UGSAS-GU)
P22: Akash Chandela (UGSAS-GU)
P23: Dina Istiqomah (UGSAS-GU; Gadjah Mada University)
P24: Kanthi Arum Widayati (Bogor Agricultural University)
P25: Yuta Shintaku (Kyoto University)
P26: Aru Toyoda (Kyoto University)

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

-PART 1INTERNATIONAL SYMPOSIUM ON ANIMAL PRODUCTION
AND CONSERVATION FOR SUSTAINABLE DEVELOPMENT

2018
KEYNOTE SPEECHESORGANIZER:
THE UNITED GRADUATE SCHOOL OF AGRICULTURAL SCIENCE,
GIFU UNIVERSITY

Proceedings of International Symposium on Animal Production and Conservation for Sustainable Development 2018
Keynote Speech: 01

Genetic improvement in goats for sustainable production
Yoko TsukaharaInternational Goat Association
American Institute for Goat Research, Langston University

SUMMARY
Goats contribute food and economic security in the world. However, the production level is generally low, andinternal parasitism has become a major constraint. Genetic improvement is one strategy to ease the problems andachieve sustainable production. Crossbreeding programs are commonly used to improve growth, dairy, andreproductive traits as well as resistance to internal parasitism especially in developing countries although the resulting
composite breed is not always adapted to the area. A goat crossbreeding simulation model was developed to predict ant
evaluate crossbreeding production scenarios. The International Goat Association that established in 1982 to promotegoat research and development conducted an intensive study evaluating various goat projects in the world.

Introduction
Goats are well adapted to various environments
and are produced all over the world. Goat production
includes meat, milk, skin, fiber (cashmere and mohair),and manure that make significant contributions to thefood and economic security of developing countries,
especially in Asia and Africa. However, the supplies ofgoat products are often inadequate to meet the growing
demand due to its low production levels as well as
health problems.
Genetic improvement is one strategy to ease theproblems and achieve sustainable production.
Crossbreeding programs, which introduce highproduction levels of exotic gene to well-adapted
indigenous breed in harsh environments, have beencommonly used in developing regions. The traits ofgreatest importance in goat production include growthrate, milk production, reproductive performance, andresistance to internal parasitism. In general, exotic
breeds are higher in milk yields and mature weights
than the indigenous breeds, whereas indigenous breeds
generally possess high reproductive performance andparasite
resistance.
Therefore,crossbreeding
indigenous breeds with exotic breeds can be expected
to improve their productivity with maintaining theirhigh adaptability to the harsh environments.
The objectives of this paper are to provide an
overview of research of crossbreeding programs
focused on growth, dairy, and reproductive traits andresistance to internal parasitism in goats and to
introduce activities of the International Goat
Association.

example of crossbreeding programs involving
indigenous Katjang breed with German Fawn breed in
Malaysia. The objective of this program was toproduce crossbreds combined high genetic potentials of
growth rate and dairy performances of the German
Fawn and the high fecundity, survivability, andadaptability to Malaysian environments of the Katjang.
The goal of the program was to establish twocomposite breeds; the Jermasia which was a meat
breed and composed 50% of each sire and dam breed
genes and the Jermana which was a dairy breed
composed 75% of the German Fawn gene (Mukherjee
et al., 1985; Devendra, 2007).
Numerous crossbreeding programs of goats havebeen carried out in developing countries, but many ofthe programs were not successful or adapted to theareas. For instance, Jamnapari dairy goat from India
has been used extensively in Southeast Asian countries
to improve milk yield and body size of indigenous
goats, however, the large mature body size of theresulted crosses can be a disadvantage during the feed
shortage period in dry seasons. Ayalew et al. (2003)
collected data on goat production from farmers to test
the assumption that crossbreds would be more
beneficial than indigenous goats under improved
management and concluded better management ofindigenous goats could improve household welfare
without producing crossbreds. Tsukahara et al. (2011)
developed a goat crossbreeding simulation model topredict and evaluate crossbreeding production
scenarios. The model compared production efficienciesof crossbreeding systems in the tropic conditions andreported the greater production efficiency of composite
breeds (F1 and BC) than of indigenous in the intensive
dairy production scenario and greater production
efficiency and stocking rate of indigenous goats than ofcomposite breeds under the integrated goat meat andoil palm production scenario.

Examples for crossbreeding systems ingoats
Crossbreeding programs in goats typically employ
exotic breed bucks of high performance, and smallsized indigenous does to produce F1 breed, and then,backcross breeds (BC) by crossing exotic bucks with
F1 does (Hirooka et al., 1997). Figure 1 shows an2Proceedings of International Symposium on Animal Production and Conservation for Sustainable Development 2018
Keynote Speech: 01

utilizing data from a crossbreeding program involving
the exotic German Fawn and the indigenous Katjang
goats in Malaysia.

Exotic (E)Local (L)

Growth traits

F1
.5L
.5E

L

L

BC1
.75L
.25E

F2
.5L
.5E

L

F1㹶BC1
.625L
.375E

F3
.5L
.5E

Establishmentof a composite
meat breed

Growth traits including birth weight, weaning
weight, and yearling weight are of importance for meatproduction and have been used to evaluate growth
performance in goats. However, those traits could only
observe fragmentary aspects of growth from birth todeath (or culling) of goats. Instead, Tsukahara et al.
(2008a) fit the longitudinal age-weight data from thecrossbreeding program involving the exotic German
Fawn breed and the indigenous Katjang breed to
growth curve models and compared growth patterns of
F1, F2 and BC2 (75% German Fawn) does. This study
showed the maturing rate of the backcross (BC2) was
higher than those of other genotypes, while theindigenous Katjang breed required more time toachieve the mature weight than other genotypes(Figure 2). Moreover, the significant influence of the
genotype over mature weights and maturing rates werepositively related in most cases and therefore, possible
simultaneous improvement of the two growth traits bycrossbreeding were suggested (Tsukahara et al., 2008a).

BC2
.25L
.75E

BC3
.125L
.875E

Establishmentof a composite
dairy breed

Fig. 1. A schematic diagram of crossbreeding systems
between indigenous and exotic breeds.
shows inter semating.

The estimation of crossbreeding
parameters
Reliable crossbreeding parameter estimates areuseful measures for designing a sound crossbreeding
program. Crossbreeding parameters include additive
genetic effects, dominant effect (heterosis effect), andthe recombination loss or epistatic loss. Heterosis,
which is a measure of non-additive genetic effects ofboth intra- and inter-allelic origin, is usually estimatedas the amount by which the average performance of F1
breed exceeds the average of the two parents (Jain,
1982). Thus, the amount of heterosis can be estimatedfrom the difference between the averaged
performances of F1 breeds generated from thereciprocal crossing of sire and dam breeds andaveraged performances of pure parental breeds.
However, in goat crossbreeding programs indeveloping countries heterosis cannot be directly
estimated from the method mentioned above, because
reciprocal crosses have been rarely obtained (Hirooka
et al., 1997; Tsukahara et al., 2008a). That is, theperformance of exotic sire breed under the tropic
conditions remains unknown in most cases because thegermplasm of exotic breed is usually imported only
male animals and/or through frozen semen. To solve
the problem, Jain (1982) proposed an alternative
procedure that estimates the effect of heterosis using
the expectations of the mean performance of various
grades of crossbreeding groups. Jakubec and Hyánek(1982) described another solution for estimation ofheterosis using the magnitude of means of four types ofcrossbred populations (F1, F2, BC1, BC2) exceeding themean value of the additive gene contribution of boththe parental populations to the given generation mean.
Kahi et al. (2000) pointed out that the merit ofcrossbred genotypes can be predicted by extrapolating
the existing crossbreeding parameters. Hirooka et al.
(1997) reported a method to estimate crossbreeding
parameters on growth performance applying two mixed
models; the Dickerson model (Dickerson, 1969, 1973)
and the Kinghorn model (Kinghorn, 1980, 1983),

Dairy traits
Milk production is one of the most important
products in goats. Goat breeding, for milk productionin the United States and European countries, has been
mainly conducted within-breed selection of pure breeds(Dubeuf and Boyazoglu, 2009). In contrast,
crossbreeding to improve milk production of goats has
been often implemented in developing countries under
the tropics. Lee et al. (1975) reported that milk
performances such as milk production and lactation
periods of F1, BC2 (75% Saanen), and BC3 (87.5%
Saanen) were higher and longer than those of the
Korean Native goats, whereas milk fat percentages ofcrossbreds were lower than that of the Native, further,
there was no significant difference between purebred
Saanen and BC3 in milk production, lactation period,and milk fat percentage. Montaldo et al. (1995)
compared dairy performance of high-grade ( 15/16),
and low-grade ( 7/8 of any breed) crosses of Alpine,
Granadina, Nubian, Saanen, and Toggenburg sires withlocal Mexican goats in northern Mexico, and reported
that Alpine, Saanen and Toggenburg crosses hadgreater milk production and lactation length than
Granadina and Nubian crosses or local does but nodifferences between high- and low-grade goats. Prasadand Sengar (2002) studied milk yield and compositionof the dual purpose Barbari breed and its crosses with
the Jamnapari, Beetal (milk breeds), and Black Bengal(meat breed) in India, and concluded that the breed
effect was significant on milk yield and composition;
Barbari sired Beetal crosses produced significantly
more milk than the Barbari and the other crosses, andthe milk fat percentage of Barbari sired Black Bengal
crosses was significantly higher than the others.
Also, Ruvuna et al. (1995) investigated effects ofcrossbreeding on lactation curve parameters of Wood’s

3Proceedings of International Symposium on Animal Production and Conservation for Sustainable Development 2018
Keynote Speech: 01

Anous and Mourad (1993) characterized the
Alpine milk breed, the Rove meat breed, and their firstcross performance in France and reported that effect of
the breed was not significant on fertility but highly
significant on abortion and prolificacy of does. In
addition, they showed that heterosis was positive andsignificant for prolificacy and fertility, while not for
abortion rate. Montaldo et al. (1995) reported that litter
size was greater in low-grade ( 7/8 of exotic breed)
Saanen and Nubian crosses and the lower values were
found in high-grade (15/16 of exotic breed)
Toggenburg crosses, furthermore, litter weight means
were significantly larger in high-grade Alpine andlower in high-grade Granadina. Tsukahara et al.
(2008b) showed that crossbred goat groups showed
higher performances in total litter weights and average
litter weights than the indigenous breed, while litter
size of crossbred goat groups was lower than that of theindigenous breed.
Though conception rates, service period, and litter
size are generally treated as female maternal traits,
influences of the sire of the fetus on these traits havebeen reported in cattle, sheep, swine, and mice (Kadryand Afifi, 1982/1983). Tsukahara et al. (2008b)
estimated crossbreeding parameters to investigate theinfluence of parental genotypes and paternal heterosis
on litter traits, regarding litter size, total litter weight,
and average litter weight as traits of the litter using datafrom a crossbreeding experiment in Malaysia (Table 1).
The results revealed that the genotype of sire had asignificant and negative effect on litter size, andpaternal heterosis had a significant and negative
influence on total litter weight and average litter weight.

100

Degree of maturity (%)

80
60
Katjang
40
20
0
0

25

50

75
Age in week

100

125

Fig. 2. Degree of maturity from time of birth to over 125
weeks of age by genotype in the Bertalanffy model.

equation ( Yt

at b e  ct ), fitting data derived from acrossbreeding program conducted in Kenya involving
two indigenous goat breeds (East African and Galla)
and two exotic breeds (Toggenburg and Anglo Nubian).
As a result, they reported that all lactation curveparameters (a, initial average milk yield: b, the rate ofincrease to peak production: c, the rate of decline after
peak production) differed among the breed groups; theindigenous breeds ranked lowest for parameter a andhighest for c, and the four-way cross was superior forpeak yield, total lactation yield and the value of b.
However, most of the genetic improvement
programs for dairy performance of indigenous goats
have been failures and unsustainable (Devendra, 2007;
Dubeuf and Boyazoglu, 2009). Serradilla (2001)
reviewed reported experiments on the raising of highyielding breeds of goats under suboptimal conditions indifferent areas of the world, concluding some general
reasons for impairing the success of these programs; 1)
milk yield of F1 does are intermediate between those of
the parental breeds or, in some cases, closer to theexotic breed, nevertheless the effect of heterosis formilk yield has seldom been reported, 2) composition ofmilk, such as protein, fat, and total solids from F1 does
are either similar to those of local breeds orintermediate between those of the parental breeds, 3)
backcrossed does generally do not show clear
differences with F1 for dairy traits, while does with ahigher proportion of exotic genes (15/16) produced
more milk than those with a lower proportion (7/8), 4)
F2 does show worst dairy performances than F1 andbackcrosses, and F3 does are similar to those of local
breeds.

Table 1

Estimations of crossbreeding parameters for litter traits (Tsukahara etal. 2008b)
Parameters1)
P

g

M

Total litter weight-0.212 **

0.23

0.158

1.712

Average litter
weight0.475 ****

0.984 **

-0.007-0.21 *
-0.164hPMg , addirtive paternal breed effect; g , additive maternal breedPeffect; h , paternal heterosis effect*
P < 0.05, ** P < 0.01

**

1) P

Resistance to internal parasitism
Goats have peculiar features in adaptability suchas anatomical, morphological, and physiological
adaptation, and this unique character allows their wide
distribution through the hot and cold climates(Devendra, 2007). This implies that high adaptability
such as heat tolerance and resistance to diseases andparasites of indigenous goats in tropic regions are
significantly related to their survivability. For example,
Al-Busaidi et al. (2008) investigated significantly
increased white blood cell counts and immunoglobulinlevels of Dhofari goats, indigenous to Oman, during
the summer, suggesting high resistibility of the breed
under hot environment.
Internal parasitism has become a major constraint
to small ruminant production all over the world due towidespread anthelmintic resistance. Ricordeau (1981)
reported that the survival rate of progeny between


Reproductive traits
Reproductive traits are a major determinant of
productivity and economic viability (Mourad, 1994;
Mellado et al., 2006). In particular, the reproductive
rates of goats are undoubtedly the highest among thedomestic ruminant species and the length of thebreeding season shows distinct differences between
breeds, hence, the reproductive performances of goats
are considered more important than that of otherdomestic ruminant species. Despite the low heritability
and viability of the reproductive performance, thegenetic improvement of those traits throughcrossbreeding has still been expected.

gLitter size

4Proceedings of International Symposium on Animal Production and Conservation for Sustainable Development 2018
Keynote Speech: 01

bucks selected for resistance to internal parasites(Haemonchus contortus) and untested does was muchhigher (71%) than that of controls (33%). Pralomkarn
et al. (1997) reported that Thai native goat kids hadgreater resistance to H. contorts than crossbred (50%Thai native × 50% Anglo Nubian) kids although live
weight gain was higher in crossbred kids than native
kids. Fecal egg count is commonly used as an indicatorof haemonchusis, and its heritability has been reported
between 0.13 (75% Kiko × 25% Boer; Thomas et al.,2016) and 0.32 (crossbred cashmere-producing goats;
Vagenas et al., 2002). Tsukahara et al. (2016)concluded that selection for resistance to H. contortus
did not advisory affected growth performance of malegoat kids in a central performance test in the southcentral US.

Activities of
Association

theInternational

6) Dickerson, G. E. (1973) Inbreeding and heterosis
in animals. In: Proceedings of the Animal
Breeding and Genetics Symposium in Honor of Dr.
J. L. Lush. American Society of Animal Scienceand American Dairy Science Association, 54-77.
Champaign, IL.
7) Dubeuf, J.-P., and Boyazoglu, J. (2009) Aninternational panorama of goat selection andbreeds. Livest. Sci., 120:225-231.
8) Hirooka, H., Mukherjee, T. K., Panandam, J. M.,
and Horst, P. (1997) Genetic parameters for
growth performance of the Malaysian local goatsand their crossbreds with the German (improved)
Fawn goats. J. Anim. Breed. Genet., 114:191-199.
9) IGA (International Goat Association). (2014)
Scaling-up successful practices on sustainable propoor small ruminant development. (Eds) J.-P.
Dubeuf, B. A. Miller, D. Bhandari, J. Capote, J.-M.
Luginbuhl. Little Rock, Arkansas, USA, pp 436.
10) Jain, J. P. (1982) Note on the estimation ofheterosis when the performance of parents is notknown. Indian J.Anim. Sci., 52:339-341.
11) Jakubec, V., and Hyánek, J. (1982) Quantitative
analysis of components of hybridization. Livest.
Prod. Sci., 9:639-651.
12) Kadry, A. E. H., and Afifi, E. A. (1982/1983)
Effect of the buck on number of services perconception, service period and litter size at birth in
Bauscat Rabbits. Anim. Reprod. Sci., 5:219-222.
13) Kahi, A. K., Thorpe, W., Nitter, G. and Baker, R.
L. (2000) Crossbreeding for dairy production in
the lowland tropics of Kenya. II. Prediction of
performance of alternative crossbreeding strategies.
Livest. Prod. Sci., 63:55–63.
14) Kinghorn, B. (1980) The expression of‘Recombination Loss’ in quantitative traits.
Zeitschrift Fur Tierzuchtung Und
Zuchtungsbiologie, 97:138-143.
15) Kinghorn, B. (1983) Genetic effects incrossbreeding. III Epistatic loss in crossbred mice.
Zeitschrift Fur Tierzuchtung Und
Zuchtungsbiologie, 100:209-222.
16) Lee, K. W., Choi, K. S., Tak, T. Y., Sul, D. S., andLee, K. M. (1975) Improvement of Korean Native
goats by grading-up with Saanen: II. Change on
milk performance of crossbreds between Korean
Native goats and Saanen. Korean J. Anim. Sci.,
17:50-57.
17) Mellado, M., Valdéz, R., García, J. E, López, R.,
and Rodríguez, A. (2006) Factors affecting the
reproductive performance of goats under intensive
conditions in a hot arid environment. Small Rumin.
Res., 63:110-118.
18) Montaldo, H., Juárez, A., Berruecos, J. M., andSánchez, F. (1995) Performance of local goats andtheir backcrosses with several breeds in Mexico.
Small Rumin. Res., 16:97-105.
19) Mourad, M. (1994) Estimation of genetic andphenotypic parameters of some reproductive traitsof African Common goats in Rwanda. Small
Rumin. Res., 15:67-71.
20) Mukherjee, T. K., Samudram, A. R., and Sivaraj, S.
(1985) Milk composition of local and F1 (local

Goat

The International Goat Association (IGA)
promotes goat research and development for the
benefit of humankind, to alleviate poverty, to promote
prosperity and to improve the quality of life in theworld. The main activities include 1) organizing an
International Conference on Goats (ICG) every four
years, 2) publishing Small Ruminant Research, thepremier journal of goat and sheep research, and 3)
promoting knowledge exchange to link research,
production, marketing, and consumption of goatproducts. The IGA conducted an intensive study
entitled “Scaling-Up Successful Practices on
Sustainable Pro-Poor Small Ruminant Development”
from 2011 to 2012 with support from the International
Fund for Agricultural Development and reported
success and failure of various goat projects in theworld (IGA, 2014). The IGA is serving on the advisory
council of the FAO and OIE coordinated program
entitled “Global Strategy for the Control and
Eradication of PPR (Peste des Petits Ruminants)” until
the year 2030. IGA is holding the 13th International
Conference in Eger, Hungary in September 2020.

References
1) Al-Busaidi, R., Johnson, E. H, and Mahgoub, O.
(2008) Seasonal variations of phagocytic response,
immunoglobulin G(IgG) and plasma cortisol levels
in Dhofari goats. Small Rumin. Res., 79:118-123.
2) Anous, M. R., and Mourad, M. M. (1993)
Crossbreeding effects on reproductive traits ofdoes and growth and carcass traits of kids. Small
Rumin. Res., 12:141-149.
3) Ayalew, W., Rischkowsky, B., King, J. M., andBruns, E. (2003) Crossbreds did not generate more
net benefits than indigenous goats in Ethiopian
smallholdings. Agric. Syst., 76:1137-1156.
4) Devendra, C. (2007) Goats: Biology, production
and development in Asia. Academy of Science,
Kuala Lumpur, Malaysia.
5) Dickerson, G. E. (1969) Experimental approaches
in utilizing breed resources. Anim. Breed.
Abstracts., 37:191-202.

5

Proceedings of International Symposium on Animal Production and Conservation for Sustainable Development 2018
Keynote Speech: 01

female × improved German Fawn male) goats.
Malaysian Appl. Biol., 14:100-103.
21) Pralomkarn, W., Pandey, V. S., Ngampongsai, W.,
Choldumrongkul, S., Saithanoo, S., Rattaanachon,L., and Verhulst, A. (1997) Genetic resistance of
three genotypes of goats to experimental infection
with Haemonchus contortus. Vet. Parasitol.,
68:79-90.
22) Prasad, H. and Sengar, O. P. S. (2002) Milk yieldand composition of the Barbari goat breed and its
crosses with Jamunapari, Beetal and Black Bengal.
Small Rumin. Res., 45:79-83.
23) Ricordeau, G. (1981) Genetics: Breeding plans. In:
Goat Production. (Gall C. ed.) 111-169. Academic
Press. London.
24) Ruvuna, F., Kogi, J. K., Taylor, J. F., and Mkuu, S.
M. (1995) Lactation curves among crosses ofGalla and East African with Toggenburg andAnglo Nubian goats. Small Rumin. Res, 16:1-6.
25) Serradilla, J. M. (2001) Use of high yielding goat
breeds for milk production. Livst. Prod. Sci.,
71:59-73.
26) Thomas, C. L., Lamberson, W. R., Weaber, R. L.,
Wilbers, L. S., Wuliji, T., Caldwell, J. D., andShanks, B. C. (2016) Genetic parameters forinternal parasite resistance, reproduction, andgrowth traits in a closed line of Kiko × Boer goats
divergently selected for internal parasite resistance.
Sheep and Goat Res. J., 31:30-37.
27) Tsukahara, Y., Chomei, Y., Oishi, K., Kahi, A. K.,
Panandam, J. M., Mukherjee, T. K., and Hirooka,
H. (2008a) Analysis of growth patterns inpurebred Kambing Katjang goat and its crosses
with the German Fawn. Small Rumin. Res., 80:815.
28) Tsukahara, Y., Choumei, Y., Oishi, K., Kumagai,
H., Kahi, A. K., Panandam, J. M., Mukherjee, T.
K., and Hirooka, H. (2008b) Effect of parental
genotypes and paternal heterosis on litter traits incrossbred goats. J. Anim. Breed. Genet., 125:8488.
29) Tsukahara, Y., Oishi, K., and Hirooka, H. (2011)
Development and application of a crossbreeding
simulation model for goat production systems intropical regions. J. Anim. Sci., 89:3890-3907.
30) Tsukahara, Y., Gipson, T., Hart, S. Dawson, L.,
Wang, Z., Puchala, R., Sahlu, T., and Goetsch, A.
L. (2016) Progress in resistance to internalparasitism and growth performance of Boer, Kiko,and Spanish goat kids through selection in acentral sire test. In: Proceedings of the 12th
International Conference on Goats, Antalya,
Turkey, pp.131.
31) Vagenas, D., Jackson, F., Russel, A. J. F.,
Merchant, M., Wright, I. A., and Bishop, S. C.
(2002) Genetic control of resistance to gastrointestinal parasites in crossbred cashmereproducing goats: responses to selection, genetic
parameters and relationship with production traits.
Anim. Sci., 74:199-208.



6

Proceedings of International Symposium on Animal Production and Conservation for Sustainable Development 2018
Keynote Speech: 02

Animal welfare and behaviourShigeru Ninomiya
Faculty of Applied Biological Sciences, Gifu University, JapanSUMMARY
When we consider animal welfare, Freedom to express normal behaviour, which is one of Five freedoms being
recognized as a principle of animal welfare in the world is the most challenging issue. Applied Ethology has beenworking on the task but we can not understand animal behaviour completely. In this lecture I will talk about therelationship between animal welfare and behaviour. There are two theme, one is ‘Is performing behaviours important
for animals?’ and the other is Environmental enrichment. I will also introduce our study regarding environmental
enrichment for fattening beef cattle, which has been conducted for investigating their motivation to perform grooming
behaviour at Minokamo Farm of Gifu university.

Introduction

Performing behaviour is also important for the other

Animal welfare in keeping animal has been a global

aspects of Five freedoms. For example, searching for

issue and many countries have to deal with the issue.

resources will enable animal to get foods or water, andFive freedoms is well recognized as a principle of animal

this meets Freedom from hunger and thirst. Moving to a

welfare and includes five aspects; Freedom from hunger

more comfortable place will help animals to keep theirand thirst, Freedom from discomfort, Freedom from pain,

homeostasis, and this meets Freedom from discomfort.

injury or disease, Freedom to express normal behaviour

Performingand Freedom from fear and distress (Farm Animalgrooming) will increase animal’s physical health, andWelfare Council, 1992).

this meets Freedom from disease. Social behavior will

When we keep animals in artificial environments,

improve affinity with peers, and this meet Freedom from

Freedom to express normal behaviour is the mostfear and distress (Table 1).

maintenancebehavior(e.g.

resting,challenging issue. Applied Ethology has been working
Table 1 Five freedoms suggested by FAWC (1992) andanimal behaviours to ensure them.

on the task, and the International Society for Applied
Ethology holds scientific meeting every year. Although
animal behavior is not perfectly explained by scientists,
Applied Ethology and the society has revealed important
knowledge related to behavioral restrictions in intensive
husbandry systems (Mason & Burn 2011). In Japan,
Japanese Society for Applied Animal Behaviour deals
with this issue.

Five Freedoms

Animal behaviorr†

Freedom from Hunger andThirstSearching for resources

Freedom from Discomfort

Moving to a more
comfortable place

Freedom from Pain, Injury
or Disease

Maintenance behaviour

Freedom to Express Normal Normal Behaviour
Behaviour

Is performing behaviours important foranimals?

Freedom from Fear andDistress

In artificial environments, animals are sometimes
restricted to perform behaviours. Why does behavioural

Social behaviour† Environmental enrichment is conducted to stimulate
behaviours.

restriction decrease animal welfare? Animal behavioral
needs will be generated by external stimuli such as

Environmental enrichment

stressors deriving from their environment or the methodof animal care, or some internal factor in farm animals.

Environmental

enrichment

is

one

of

animalThis means that behavioral restriction would induce

management methods which enables animals to perform

maladaptation to stressors or chronic stress (Ninomiya

an adequate behavior for coping with stressors or satisfy2014).

their behaviour needs.

7Proceedings of International Symposium on Animal Production and Conservation for Sustainable Development 2018
Keynote Speech: 02

In zoo, environmental enrichment has been more

Cattle’s behaviour were recorded using CCD cameras

popular in recent years. Environmental enrichment isand a digital video recorder and from the movie wealso cited in modern intensive husbandry. In laying hens,

observed their behaviour using a continuous behaviouralan enrichment cage, which includes some devices forrecording. I will talk about the effect of the grooming

nesting, dust baths and perching are usually provided has

devicebeen developed (Appleby & Hughes 1995). The devices(self-grooming, social grooming) in fattening beef cattle

enable laying hens to perform pre-egg-laying behavior,in this lecture.

onperforming

grooming

behaviour

dust-bathing and perching. In a pig’s housing system,
beddedsystemsareacceptedasConclusion

environmental

enrichment for pig behavioral needs for rooting (van deThere have been many issues around animal welfare yet

Weerd & Day 2009).

and Freedom to express normal behaviour is the most

In cattle, devices for self-grooming were used (Pelleychallenging issue. I have been studying animal welfare

et al. 1995; Wilson et al. 2002). In fattening cattle farms,

and behaviour, and the study introduced in this lecture is

it is difficult to clean each cattle’s body or increase

one part of my study. Animal behavior is not perfectly

frequency of muck managements because productive

explained by scientists and therefore I hope that future

HI¿FLHQFLHV SHU XQLW DUHD DQG ODERU DUH VRXJKW IRUstudy will clarify it.

economic rationality. In the case where animals are dirty

Acknowledgement

because of poor management of animal feces or urine,
setting a device to enable animals to perform

I am grateful for conducting the study of environmental

self-grooming behavior in housing will be a valid

enrichment in fattening beef cattle by students oftreatment for their welfare.

Laboratory of Animal welfare and behaviour, Gifu

Our laboratory has been investigating the effect ofgrooming

deviceinfattening

beef

cattleUniversity, Japan.

asenvironmental enrichment in terms of their motivation toReference

perform grooming behaviour at Minokamo Farm of Gifu

Appleby MC, Hughes BO. (1995) The edinburgh
PRGL¿HGFDJHIRUOD\LQJKHQV%ULWLVh Poultry Science36, 707–718.
Farm Animal Welfare Council (1992) FAWC updates the¿YHIUHHGRPV9HWHULQDU\5HFRUG
Mason GJ, Burn CC. (2011) Behavioural Restriction. In:
Appleby MC, Mench JA, Olsson IAS, Hughes BO(eds), Animal Welfare, pp. 98–119. 2nd edn. CABI,
Oxfordshire
Ninomiya S (2014) Satisfaction of farm animal
behavioral needs in behaviorally restricted systems:
Reducing stressors and environmental enrichment.
Animal Science Journal, 84:634-638.
Pelley MC, Lirette A, Tennessen T. (1995) Observations
on the responses of feedlot cattle to attempted
environmental enrichment. Canadian Journal ofAnimal Science 75, 631– 632.
van de Weerd HA, Day JEL. (2009) A review ofenvironmental enrichment for pigs housed in intensive
housing systems. Applied Animal Behaviour Science
116, 1–20.
Wilson SC, Mitlohner FM, Morrow-Tesch J, Dailey JW,
McGlone JJ. (2002) An assessment of several potential
enrichment devices for feedlot cattle. Applied Animal
Behaviour Science 76, 259–265.

university. The device was combined by two brush. One
brush measuring 50 cm in length and 10 cm in width wasarranged together with a second brush in an inverted
L-shaped pattern (Albert Kerbl, Buchbach, Germany).
One brush was fixed horizontally about 130 cm from thefloor and the other brush was attached to the wall of thepen.

Fig.1 Grooming device and fattening beef cattle.

8

Proceedings of International Symposium on Animal Production and Conservation for Sustainable Development 2018
Keynote Speech: 03

Maternal antibody transfer into eggs and new-born chicks in chicken and quail
Atsushi Murai
Graduate School of Bioagricultural Sciences, NAGOYA University
SUMMARY
In avian species, maternal immunoglobulin Y (IgY) is transferred into egg yolks of maturing oocytes to protect the
new-born chicks against pathogenic attacks, but the molecular basis of IgY transfer is still unknown. To determine thestructural requirements of IgY for selective transfer into avian egg yolks, recombinant wild-type IgY-Fc (WT) and its
mutants were synthesized, and their uptakes into the egg yolks of chicken and quail were determined. The substitutionof Y363 at the CX3 domain to alanine abolished the IgY-Fc uptake into egg yolks. Interestingly, substitution of G365 foralanine elevated IgY-Fc uptake. Characterization of several candidate IgY receptors responsible for maternal antibody
transfer was performed. Our results emphasize the existence of a selective maternal antibody transport system
recognizing the CX3 domain of IgY, which raises the possibility that an IgY with high transport ability might beengineered by genetic manipulation.

Introduction

the Fc domains of IgY. In addition, several candidate

In avian species, IgY, the counterpart to mammaliangenes responsible for antibody transfer into egg yolks

IgG, is transferred from the maternal blood circulation towere characterized by gene expression analyses,

the embryonic or new-born chick circulation. The

immunohistochemistry and binding assay to IgY-Fc

process of avian maternal IgY transfer is divided into

mutants.

two steps: the first step is the transfer from the maternal

9+blood to the egg yolks of developing oocytes, and the9/second step is the transfer from the egg yolks to theembryonic circulation through the yolk sac membrane in&X

the developing eggs. The second step relies on an IgY-Fc&/&X

receptor called FcRY (West et al., 2004). In the first step,&Xhowever, the relevant receptor of the maternal IgY&X)F

&X

transfer is still unidentified
An understanding of the structural requirements of

&X

<
<

,J<

IgY and for selective transport into egg yolks is ofFig. 1. The domain structure of chicken IgY (left) andconformation of IgY-Fc (right).

importance for gaining insight into the molecular basis ofmaternal IgY transfer. In our previous study, the intact

Material and Method

chicken IgY or its Fc fragment was incorporated into

1. Measurement of intravenously-injected recombinantyolks of quail more effectively than Fab and F(ab’)2

IgY-Fc concentrations in egg yolks

fragments (Kitaguchi et al., 2008). The IgY heavy chain

To synthesize chicken and quail IgY-Fc mutants, theconsists of four constant domains, and the Fc fragment
mainly contains two constant domains on the C-terminal,

cDNA encoding the IgY X-heavy chain was isolated
from a chicken and quail splenocyte cDNA library. The

the CX3 (C upsilon 3) domain and the CX4 domain (Fig.

gene encoding the Fc regions was isolated by PCR from1). Thus, the CX3 and CX4 domain are required forthe template cDNA, and the PCR product was ligatedselective IgY transport into egg yolks.
To elucidate mechanism of maternal IgY transfer in

His tag. The constructed expression vector was used tobirds, we investigated critical amino acid residues

synthesize recombinant wild-type chicken and quail

required for efficient IgY transport into the egg yolks by

IgY-Fc (designated WT). The amino acid residues

mutational analyses of selected residues located along

located on the CX3 domain of IgY-Fc were individually

into the mammalian expression vector, with a C-terminal

9

Proceedings of International Symposium on Animal Production and Conservation for Sustainable Development 2018
Keynote Speech: 03

substituted for alanine or other amino acid residues by

revealed that FcRY expresses in granulosa cell layer andsite-directed mutagenesis (Fig. 1). The generated WT

theca layer of ovarian follicle in chicken, although its

and mutant constructs were then transiently transfected

expression level was lower than that of yolk-sac

into CHO-S cells, and the expressed WT and mutants

membrane.

were purified by His-affinity columns.

preferentially bound to WT compared with Y363A

Pull-down

assay revealed

that

FcRY

mutant, which showed a similar pattern of WT and

The purified proteins were labeled with digoxigenin.

Y363A transfer into egg yolks.

The regularly laying chicken and quail were injected

Another

intravenously with native and recombinant IgY-Fc or its

candidategene,

LRP2L,

washighlymutants. Uptakes of the injected IgY-Fc into egg yolks

expressed in ovary compared with other body tissues

were measured by ELISA.

(liver, adipose tissue etc.). Microscopic analysis of

2. Characterization of candidate IgY receptor genes forovarian follicles showed that LRP2L specificallymaternal antibody transfer

expressed in oocyte plasma membrane within ovarian

FcRY gene: To obtain a specific antibody against FcRY,

follicular tissues. These results suggest that LRP2L

rabbit was immunized with recombinant chicken FcRY.

specializes in incorporation of egg yolk precursor in

The obtained polyclonal antibody against FcRY was usedovary. Binding analyses of both FcRY and LRP2L

for analysis of FcRY expression in maternal ovary andcontinues to be investigated.

yolk-sac membrane of the developing embryos. Binding140

ability of IgY-Fc mutants to FcRY was measured by% of uptake (vs.WT)

pull-down assay
Low-density lipoprotein receptor-related 2-like (LRP2L)
gene: LRP2L gene was selected as a candidate gene byprotein-protein interaction analysis between IgY andisolatedovariansheetsincludingoocyteplasmamembrane. Body tissue expression pattern of LRP2L
gene was characterized by reverse transcribed-PCR.

120
100

*

80
60
40

* *

*
*

*

20

*
*

*

*

0

*

ND

Ovarian tissue localization and intensity of LRP2L
expression was analyzed using polyclonal antibody.

Fig. 2. IgY-Fc mutants with single alanine substitution
and their uptakes into egg yolks of quail. *P<0.05 (vs.
WT). n = 20 in WT and 4 or 5 in mutants.

Result and Discussions
1. Uptakes of IgY-Fc mutants in egg yolks
Among the 17 amino acid residues located on theConclusion

CX3/CX4 interface of chicken IgY-Fc, the substitution ofImportant amino acid residues (Y363 and G365) ofY363 at the CX3 domain to alanine (Y363A) abolishedIgY required for maternal IgY transfer into egg yolks

the IgY-Fc uptake into egg yolks (Fig. 2). The

were identified. Several candidate IgY receptor genes

comprehensive substitution of Y363 with other amino

expressing in ovarian follicles were isolated and they areacids revealed that the only uptakes of the Y363F andunder investigation.

Y363W mutants maintained the same degree of the WT,

Acknowledgement

This research was supported in part by a Grant-in-Aid(No. 16H05020) from the Japan Society for the
Promotion of Science.

suggesting the residue at 363 needs to be allocated witharomatic amino acids to maintain the high transportability.
Interestingly,

Reference

when utilized quail IgY-Fc, theWest AP, Herr AB and Bjorkman PJ (2004) The chicken
yolk sac IgY receptor, a functional equivalent of the
mammalian MHC-related Fc receptor, is a
phospholipase A2 receptor homolog. Immunity,
20:601-610.
Kitaguchi K, Osada K, Horio F and Murai A (2008)
Exclusion of polymeric immunoglobulins andselective immunoglobulin Y transport that recognizes
its Fc region in avian ovarian follicles. Vet. Immunol.
Immunopathol., 121:290-299.

substitution of G365 with alanine (G365A) or with polarand nonpolar amino acid residues increased IgY-Fc
uptake into egg yolks compared to the WT. These results
raise the possibility that an IgY with high transportability might be engineered by genetic manipulation.
2. Characterization of candidate IgY receptor gene
Western blotting and immunohistochemical analyses

10Proceedings of International Symposium on Animal Production and Conservation for Sustainable Development 2018
Keynote Speech: 04

Restoration and utilization of abandoned fields through goat grazing
Masato Yayota1,2 and Kazuya Doi31

2

Faculty of Applied Biological Sciences, Gifu University,
Education and Research Center for Food Animal Health, Gifu University,
3
The United Graduate School of Agricultural Science, Gifu University

SUMMARY
Many fields have been abandoned due to agricultural intensification over several decades in some developed countries.
If fields are not managed properly, weedy and shrubby plants encroach the land, thereby degrading the fields anddecreasing biodiversity. Goats do not have a strict preference to plant species while grazing and can survive innutritionally harsh environments; therefore, they may be considered useful in weed management. The goal of our
project was to manage abandoned fields using goats for restoring and conserving field environments. Our results
showed that: 1) long-term (five years) goat grazing clearly changed the vegetational composition, increased the numberof plant species, and improved plant diversity in an abandoned field, 2) The goats could maintain their nutritional status
and body weight by grazing in the abandoned field for five years, and 3) cost of weed management through goat grazing
was lesser or comparable to weeding by humans with a brush cutter. Thus, goat grazing is a feasible way for thesustainable management of abandoned fields.

Introduction
Agriculturalintensification

andadecreaseinagricultural population have generated many abandoned
fields in marginal areas over several decades in somedeveloped countries. In Japan, the area of abandoned
agricultural fields is more than 400,000 ha and more thanhalf of these areas are now considered unsuitable forpaddy and crop farming (MAFF 2018) due toencroachment and overgrowth of weedy and shrubby

Fig. 1 Goat grazing as a method of weed
management. Goats feed on a various plant species,
create a patchy environment, and may recoverabandoned fields in marginal agricultural areas.

plants. Abandonment of fields may degrade the rurallandscape (MacDonald et al. 2000) and increase the riskof wildfire (Osoro et al. 2007). Moreover, encroachment
and overgrowth of weedy and shrubby plants reduce

influenced by stocking rate. Long-term grazing withplant

different

biodiversity.

Thus,

appropriate

management

stocking

rates

inducesqualitative

andmeasures should be implemented to restore and utilize

quantitative changes in field vegetation, leading tothese abandoned fields.

different animal performance and affecting sustainable

Goat grazing may be a feasible way for managing

grazing in the management of abandoned fields.

these abandoned fields (Fig. 1) because goats do not

In this brief review, we present the quantitative andhave a strict preference to plant species while grazing;

qualitative dynamics of vegetation and goat performance

they feed on grasses, forbs, and shrubs. They are capablein response to different stocking rates over a 5-yearof grazing and moving in hilly and steep areas that areperiod. In addition, we discuss the economic potential of

mainlyweed management through goat grazing by comparing

consideredabandoned

fieldsasmarginal

areas.

Generally,arecomposedofsemi-natural

this

methodwithweeding

by

a

brush

cutter.

vegetation; however, this type of vegetation is often

Understanding these dynamics will facilitate the use ofpoorer in nutritional value than sown plant species.

sustainable goat grazing systems to control abandoned

Moreover, vegetation and animal performance are largely

agricultural lands and maintain biodiversity.

11

Proceedings of International Symposium on Animal Production and Conservation for Sustainable Development 2018
Keynote Speech: 04

decreased over the five years. This continuous decrease

Vegetational dynamics with goat grazingin the diet quality decreased forage intake. However,Long-term grazing with different stocking rates clearlygoats maintained their body weight over the five years at

affected botanical composition in an abandoned field.

both stocking rates. Consequently, at the stocking rates

The dominant plant species at a high stocking rate (30studied, long-term goat grazing changed the quality ofgoats/ha) changed from bamboo (Phyllostachys edulis)

vegetation in an abandoned field; however, goats could

to grass species over five years, while a dwarf-bamboo

maintain their nutritional status and body weight over(Pleioblastus

continuously

five years (Doi et al. 2018b; 2018c). Thus, the use of

dominant in the paddock with a low stocking rate (14grazing for the sustainable management of abandonedgoats/ha) throughout the five years. The number of plant

fields is feasible and does not have negative effects on

species in the summer increased from 17-18 in the first

the nutritional status of goats.

argenteostriatus)wasyear to more than 30 species in the fifth year, regardlessEconomic potential of weeding by goatsa. 2013Goat grazing is a feasible way to control fieldabandonment; however, its economic potential should be
evaluated when we promote dissemination of thisstrategy. Economical sustainability is also crucial.
Compared with weeding by humans with a brushcutter, weeding by goat grazing largely reduced theworking time for farmers or weeding workers. Moreover,weeding cost by goat grazing was comparable to orb. 2017lesser than that by humans with a brush cutter, although
the cost was varied according to grazing days andamortization period of grazing equipment such as pasture
fence (Yayota et al. 2017). The results suggested thatgoat

grazingis

economicalformanaging

field

abandonment.


Future perspectives

Fig. 2 Goats clearly alter the vegetational
composition in abandoned fields. a. before the startof goat grazing in 2013, and b. after fiver years ofgoat grazing (2017).

Since the rapid economic growth after World War II,goat population in Japan has drastically decreased due toa reduction in demand for goat meat and milk; that is, theof the stocking rate. Shannon diversity index alsogoat industry has remained stagnant over decades.

increased from approximately 2.0 in the first year to 2.5

Therefore, it is important that goats are given priority inin the fifth year at both high and low stocking rates (Doithe agricultural economy. Goats could be useful inet al. 2018a). These results suggested that goat grazing in

restoring, managing, and conserving agricultural field

an abandoned field for five years clearly changed theenvironments. As shown here, this requires an ecologicalvegetational composition, increased the number of plant

approach as well as an approach considering animal

species, and improved plant diversity (Fig. 2).

production science. In addition, its socio-economicalvalue should not be forgotten, after all, goats are an

Sustainable goat grazing

important component of livestock.

Goat grazing at various stocking rates (14-30 goats/ha),Intensification of agricultural processes and livestock

did not decrease forage biomass over time. Changes in

production is effective and inevitable for economic

the botanical composition affected the quality of the diet.

growth; however, it also negatively affects use of land

At the high stocking rate, the diet quality, which wasand other resources, as well as the biodiversity and thereflected by crude protein (CP) and neutral detergent

rural landscape. Therefore, we should consider a more

fiber (NDFom), was relatively constant during the study

comprehensive solution for maintaining agricultural

period, whereas at the low stocking rate, diet quality

12Proceedings of International Symposium on Animal Production and Conservation for Sustainable Development 2018
Keynote Speech: 04

environments.

Acknowledgements

We are grateful to Frusic Ltd. (Pres. Shoji Watanabe) for
managing the experimental site and the animals. We also
appreciate Minokamo City for preparing theexperimental site. Part of this work was funded by the
Kisogawa Canal System Control and Management
Office, Japan Water Agency.

References
Doi K, Tamiya S, Nakajima N and Yayota M (2018a)
Effect of five years goat grazing on plant diversity inan abandoned field. Grassland Science, 64 (suppl.):102. (in Japanese).
Doi K, Achiha M, Sakoda S, Kobayashi A and Yayota M(2018b) Goat grazing an abandoned field: effect on
botanical composition and animal nutrition. Japanese
Journal of Grassland Science, in press. (in Japanese)
Doi K, Tamiya S, Nakajima N and Yayota M (2018c)
Can grazing goats maintain nutritional status for five
years in an abandoned field? The proceedings of the
7th Japan-China-Korea Grassland Conference, .
MAFF, 2018. Prevention and resolution of an abandoned
field.
URL
http://www.maff.go.jp/j/nousin/tikei/houkiti/.
(accessed 29 June 2018)
MacDonald D, Crabtree J R, Wiesinger G, Dax T,
Stamou N, Fleury P, Gutierrez Lazpita J and Gibon A(2000) Agricultural abandonment in mountain areas ofEurope: Environmental consequences and policy
response. Journal of Environmental Management, 59:
47-69.
Osoro K, Garcia U, Jauregui B M, Ferreira L M M, Rook
A J and Celaya R (2007) Diet selection andlive-weight changes of two breeds of goats grazing onheathlands. Animal, 1: 449-457.
Yayota M, Doi K, Tamiya S, Matsuda H, Ohtsuka H and
Watanabe S (2017) Comparison in working time andlabor cost between weeding by goats and brush cutter.
The proceedings of the 18th Japanese Society of Goat
Research, 14-15.

13

Proceedings of International Symposium on Animal Production and Conservation for Sustainable Development 2018
Keynote Speech: 05

What we know and what we need to know about foregut-fermenting primates: a case ofproboscis monkeys in Borneo
Ikki Matsuda
Chubu University Academy of Emerging Sciences; Wildlife Research Center, Kyoto University; Japan Monkey
Centre; Institute for Tropical Biology and Conservation,
Universiti Malaysia Sabah

SUMMARY
Today, habitat destruction and poaching threaten nearly half of the free-ranging primate species with extinction. Hence,
conservation programs have become integral aspects of zoological management, though studies in captivity haverevealed that foregut fermenting primates such as colobines are historically difficult to maintain healthy in captivity, andthat they have shorter lifespans than free-ranging individuals. Proboscis monkeys (Nasalis larvatus), endangered andendemic to Borneo, are the largest foregut fermenting colobines. They are notoriously difficult to maintain and breed in
captivity. On the other hand, knowledge about the ecology of free-ranging proboscis monkeys is also incomplete
because most studies have been conducted in mangrove and peat swamp forests from boats. However, by the 2000s, theattention raised for these unique monkeys had gradually attracted more researchers devoting their academic pursuits tothe further understanding of the species, with studies still predominantly focused on their socio-ecology, but extending
to genetic analyses, and other novel topics about their digestive capabilities and the sexual selection of enlarged noses. I
summarized the findings from our proboscis monkey long-term project in Sabah, Borneo, Malaysia and discussed theconservation implications of our results

Background
Proboscis monkeys (Nasalis larvatus) have uniquelyenlarged noses that are prominent adornments, especiallyfor males (Koda et al. 2018), with enlarged andsacculated fore-stomach (Matsuda et al. 2015) (Fig. 1).
However, for decades, knowledge about endangeredproboscis monkey had been gleaned from limited
information of their behavior from boat surveys along
riverbanks where they rest in the early mornings and lateevenings. This was due to the inhospitable swampy
habitats they live in, which made them near impossible
to track once they move into inland forests (Fig. 2). This
resulted in patchy knowledge about the species’ ecology.

Fig. 1 They are large, sexually dimorphic, andprimarily arboreal, colobine primates. Adult males
have the largest body size among colobine species (ca.
20 kg), whereas adult females are about half theweight of males.

14

Proceedings of International Symposium on Animal Production and Conservation for Sustainable Development 2018
Keynote Speech: 05

We pioneered the first full-day continuous
observations of proboscis monkeys by tracking them onfoot in riverine forests in Sabah, Borneo, Malaysia. This
long-term effort which began in 2005 led to the firstsystematic study of proboscis monkeys, revealing
comprehensive information about their feeding andranging behaviors; as well as novel findings about theirincredible digestive strategy of rumination, the first
documented among primate species (Matsuda et al.
2011).
Understanding the natural diet of species may
provide useful information that can contribute tosuccessful captive maintenance. A common problem
experienced with captive foregut-fermenting primate

Fig. 2 Proboscis monkeys are endemic to the island of(colobine) diets is that they are deficient in fiber and

Borneo. They are restricted mainly to swampy habitats

therefore highly digestible. This may contribute toalong waterways such as mangrove, peat swamp andgastrointestinal disorders often observed in zoos. Indeed,riverine forests. Those habitats make continuous

some colobines like proboscis monkeys are notoriously

observations on foot very difficult.

difficult to maintain and breed in captivity.

period in the Kinabatangan floodplain; however, certain

Here, I reviewed our in-situ and ex-situ

areas within the proboscis monkey’s range are still underproboscis monkey project to understand what we know

threat and need to be protected to ensure the species’

and what we need to know about this endangered species

long-term survival (Matsuda et al. in press). Lastly, what

for their long-term conservation.

we currently know about the overall population status of
the species across its range (Borneo) is still based largely

Population status

on “guesstimates” from some 2-3 decades ago. Therefore,

Proboscis monkeys are classified as ‘Endangered’further Borneo-wide population surveys will be helpful

according to the IUCN Red List of Threatened Species

in clarifying the species’ endangered status.

(IUCN, 2008), and listed under CITES Appendix I.
Many proboscis monkey populations were formerly

Diet and digestive physiology“naturally protected” by the fact that they live in swampy

Proboscis monkeys are the largest colobines, with a diet

habitats, which are generally less threatened by humanconsisting of various proportions of leaves (38%–73%),

development compared to dry land forests due to theirfruits (11%–50%), and flowers (3%–8%) (Bennett andrelative inaccessibility (Nowak 2012). However, in

Sebastian 1988; Boonratana 2013; Matsuda et al. 2009;

recent decades, modern technology made it increasingly

Yeager 1989). Our study on their dietary choices

easy to meliorate these habitats and reclaim swamplandconsidering a variety of nutritional and mechanicalby filling it with compressed soil, so swamps quickly

factors with diet digestibility found that the preferredbecame a cheap source of land available for humanleaves of proboscis monkeys not only contain more

exploitation, including industry and human settlements

protein and less fibre but are also less tough and more

(Lhota et al. in press-a). Many populations are now

digestible than the alternatives (Matsuda et al. 2017).

found scattered in small patches of suitable habitats (Sha

Proboscis monkeys are the only colobine

et al. 2008).
In

our

study

conducted

inthespecies in which an apparent rumination of stomach

lower

contents has been observed under free-range conditions

Kinabatangan region, Sabah, Malaysia, the relatively(Matsuda et al. 2011). However, displaying marker

stable population was found within 10 years (2004-2014)

excretion patterns using fecal marker concentrations in

because there were only minor losses of forest along thefour captive proboscis monkeys with a comparison ofrivers where proboscis monkeys are generally found.

that in cattle, showed the separation of the solute and theThese reduced losses were the result of increased

different-sized particle markers, observed in cattle, didprotection measures for these habitats over that 10-year15

Proceedings of International Symposium on Animal Production and Conservation for Sustainable Development 2018
Keynote Speech: 05

the National Geographic Society (#9254-13 to IM), theWildlife Reserves Singapore (Exp14/10 to IM), theInamori Foundation (to IM), JST CREST 17941861(#JPMJCR17A4) and JSPS Core-to-Core Program (to S.
Koshima).

not occur in proboscis monkeys; all markers excreted
together at mean retention times of ca. 40 h (Matsuda etal. 2015). This indicates that the digestive physiology ofproboscis monkeys is different from that of ruminantswith typical characteristics. This may be the reason why

Reference

the “suggestive rumination” is only a facultative strategy

Bennett, E.L., Sebastian, A.C., 1988. Social organizationin this species.

and ecology of proboscis monkeys (Nasalis larvatus)in mixed coastal forest in Sarawak. International

Captive study

Journal of Primatology 9, 233-255.

An approach to obtain information relevant for the

Boonratana, R., 2013. Fragmentation and its significanceimprovement of diets in captive proboscis monkeys thaton the conservation of proboscis monkey (Nasalis

are difficult to maintain healthy, is to compare thelarvatus) in the lower Kinabatangan, Sabah (Northnutrient composition of feces from free-ranging and

Borneo), In Primates in Fragments: Complexity andcaptive individuals. In theory, fecal material can be

Resilience. eds L.K. Marsh, C.A. Chapman, pp.

considered a proxy for diet intake integrated over a459-474. Springer, New York.

certain period of time. We found that feces from

Koda, H., Murai, T., Tuuga, A., Goossens, B., Nathan, S.,

free-ranging monkeys contained more fiber and less

Stark, D.J., Ramirez, D.A.R., Sha, J.C.M., Osman, I.,

metabolic fecal nitrogen than those from captive ones,

Sipangkui,indicating a less digestible diet in the wild. Our results
suggest that free-ranging proboscis monkeys basicallyS.,

Matsuda,

I.,

2018.

monkeys. Sci Adv 4, eaaq0250.

Modifying the diets of captive proboscis monkeys to

Lhota, S., Scott, K.S.S., Sha, J.C.M., in press-a. Primates

include more fiber, comparable to those of free-ranging

in flooded forests of Borneo: opportunities andones, may contribute to their health and survival.

challenges for ecotourism as a conservation strategy,
In Primates in Flooded Habitats: Ecology and

OutlookConservation. eds A.A. Barnett, I. Matsuda, K.

I reviewed our in-situ and ex-situ proboscis monkey

Nowak. Cambridge University Press, Cambridge.

project which may shed light on the establishment of a

Lhota, S., Sha, J.C.M., Bernard, H., Matsuda, I., inconstructive in-situ and ex-situ collaborative link to aid

press-b. Proboscis monkey conservation: beyond thethe management of their husbandry and possibly tonecessary

Seino,

audiovisually advertise conspecifics in proboscis

consume lower quality foods than captive individuals.

provideR.,

Nasalization by Nasalis larvatus: Larger noses

impetusforconservation

science, In Primate Research and Conservation in theandAnthropocene. eds A.M. Behie, J.A. Teichroeb, N.M.

education initiatives, which will be beneficial for theirMalone. Cambridge University Press, Cambridge

long-term conservation. However, I also need to note that

Matsuda, I., Abram, N.K., Stark, D.J., Sha, J.C.M.,

to ensure the long-term survival of proboscis monkeys, aAncrenaz, M., Goossens, B., Lackman, I., Tuuga, A.,multipronged approach would be needed according toKubo, T., in press. Population dynamics of thethe context and political situation within different

proboscis monkey Nasalis larvatus in the Lower

proboscis monkey range countries, where mechanisms

Kinabatangan, Sabah, Borneo, Malaysia. Oryx.

beyond scientific inquiry should be employed to

Matsuda, I., Clauss, M., Tuuga, A., Sugau, J., Hanya, G.,augment actions that can tilt the balance towards the

Yumoto, T., Bernard, H., Hummel, J., 2017. Factors

conservation of this unique species (Lhota et al. in

affectingpress-b).

leaf

selectionbyforegut-fermenting

proboscis monkeys: new insight from in vitro
digestibility and toughness of leaves. ScientificAcknowledgement

Reports 7, 42774.

I thank the Sabah Biodiversity Centre of the Malaysian
Government, the Sabah Wildlife Department, the Sabah
Forestry Department staff, the Kinabatangan Orangutan
Conservation Project, Danau Girang Field Centre and all
our colleagues for support. Our project was financed by
JSPS KAKENHI (#15K14605 and #21770261 to IM),

Matsuda, I., Murai, T., Clauss, M., Yamada, T., Tuuga, A.,
Bernard, H., Higashi, S., 2011. Regurgitation andremastication in the foregut-fermenting proboscis
monkey (Nasalis larvatus). Biology Letters 7,

16

Proceedings of International Symposium on Animal Production and Conservation for Sustainable Development 2018
Keynote Speech: 05

786-789.
Matsuda, I., Sha, J.C., Ortmann, S., Schwarm, A., Grandl,F., Caton, J., Jens, W., Kreuzer, M., Marlena, D.,
Hagen, K.B., Clauss, M., 2015. Excretion patterns ofsolute and different-sized particle passage markers in
foregut-fermenting

proboscismonkey(Nasalis

larvatus) do not indicate an adaptation for rumination.
Physiology & Behavior 149, 45-52.
Matsuda, I., Tuuga, A., Higashi, S., 2009. The feeding
ecology and activity budget of proboscis monkeys.
American Journal of Primatology 71, 478-492.
Nowak, K., 2012. Mangrove and peat swamp forests:
refuge habitats for primates and felids. Folia
Primatolgica 83, 361–376.
Sha, J.C.M., Bernard, H., Nathan, S., 2008. Status andconservation of proboscis monkeys (Nasalis larvatus)
in Sabah, east Malaysia. Primate Conservation 23,
107-120.
Yeager, C.P., 1989. Feeding ecology of the proboscis
monkey (Nasalis larvatus). International Journal ofPrimatology 10, 497-530.

17



-PART 1-            

INTERNATIONAL SYMPOSIUM ON ANIMAL PRODUCTION
AND CONSERVATION FOR SUSTAINABLE DEVELOPMENT

2018
GUESTS/ALUMNI PRESENTATIONSORGANIZER:
THE UNITED GRADUATE SCHOOL OF AGRICULTURAL SCIENCE,
GIFU UNIVERSITY

Proceedings of International Symposium on Animal Production and Conservation for Sustainable Development 2018
SESSION 1 - Avian Production and Reproduction -: 01

Transgenic technology: A sustainable alternative in animal production 
Asif Mahmud
Research and Development, Globe Biotech Limited, Dhaka, Bangladesh

SUMMARY
Molecular biology paved way by giving a new dimension to animal production. The emergence of transgenic
technology has widened the scope of development in case of farm animals. It is one of the important tools to meet the
future challenges for increased livestock production. The fast-growing human population increases the demand for basic
needs of human being for their survival and well-being. Transgenic animals have roles as dairy cattle as well as inbiomedical research such as gene therapy, disease resistance, drug testing, screening, toxicological screening, diseasemodel production, production of pharmaceuticals, transgenic expression of immunoglobulins, xenotransplantation, newdrug targets identification, drug development etc. Salmon is the first transgenic animal, which won US approval forfood. The US FDA has approved a chicken that has been genetically engineered to produce a drug and goats that
produce an anticoagulant. Clean meat can also be produced in the lab without raising a chicken or duck. Therefore, thegenetically engineered animals and biotechnology will play a key role in the production of pharmaceutical proteins andbring about a complete refinement in animal production by increasing the quality and quantity of production,
environment protection, maintenance of genetic diversity and overall improvement in animals’ welfare.

Introduction

competition with urban and industrial needs. Therefore,

The fast-growing human population increases theour main focus should be in obtaining high-yielding

demand for basic needs of man like food, machines etc.

varieties of desired animals, which is truly a sustainable

Being a citizen of one of the most densely populated

strategy. Several classical programs are in effect fromcountries in the world, we grew up with the thought that

relevant ministries to develop improved variety of

how the demand for basic needs can be assured. It isanimals to obtain higher yield by hybridization andquite difficult to satisfy the need of food for its 170

natural selection method. However, the success of thismillion inhabitants without some miracle. Similar

method is limited and critically depends on availability

situation exists for other countries in the world as well asof desired "super quality" among relevant species, which

for developed countries at a different perspective.

is very rare to find.

Bangladesh is one of the most densely populatedOver the ages of evolution and centuries of man-madecountries in the world. With a visionary leadership,

selection processes those "super qualities" are almost

relevant ministries nearly did that miracle. Bangladesh

used up. Therefore, it is highly unlikely to obtain abecame a food surplus country and the 5th leading"super quality" animal for improving local varieties withcountry in fresh water fish production. The meatease. Another issue is that there are no controls of human

production has also been constantly rising. As perover the natural hybridization and selection process ofYearbook of Fisheries Statistics of Bangladesh 2016-17,

any animal. With the discovery of DNA double helix by

consumptions of daily fish per person reached at 62.58 g.

Watson and Crick in 1953, the Human entered into a new

The big question is - are these achievements truly

era of knowledge - "The Genetic Edge". The art ofsustainable to satisfy the sustainable development goal

genetic science started to become useful with the(SDG). Relevant ministries have been focusing on

invention

intensive integrated farming system to increase the yield.

technology that gave the power to pick a specific genetic

Up to a certain extent it is logical but there is an obvious

element selectively from the pool of genetic materials.

saturation point; as close as we will be moving towards

Over the periods, scientists have developed technologies

that point the growth rate in yield will be nullified (Nag,to selectively transfer one gene of an animal to another,2018). Furthermore, agricultural land is in harsheven from one species to another species, and found that20ofpolymerase

chain

reaction(PCR)

Proceedings of International Symposium on Animal Production and Conservation for Sustainable Development 2018
SESSION 1 - Animal Production and Biotechnology -: 01

the transplanted gene is functioning in the new animal

can be injected into preblastoderm embryos, and cells

and executing its roles. This type of organism is calledfrom these embryos can be screened by PCR 24 h aftergenetically modified organism (GMO); intra-species andinjection. Pairs of gRNAs have been used successfully tointer-species gene transfers are called autogenic andgenerate defined deletions, and, in combination withtransgenic GMOs, respectively. Generally, GMOs are

single-strand

organisms (i.e., microorganisms, plants or animals) inCas9-mediated homologous recombination (Yu et al.,which the genetic material (DNA) has been transformed2013). This method has also been widely used within such a manner that does not happen naturally.

zebrafish. CRISPR–Cas9 reagents are injected into

Although transgenic livestock have had a high profile,zygotes, or early-stage embryos to generate mosaic F0

the practical use of these animals has been restricted toanimals. Mosaic adults are then crossed to generate

medical applications like producing pharmaceuticalhomozygous mutants (Li et al., 2016; Hwang et al.,proteins in milk, eggs etc. rather than livestock

2013; Varshney et al., 2015).

production that were originally envisioned. Use ofRodents A major advance was reported by Jaenisch andlentivirus vectors (Lois et al., 2002; Pfeifer et al., 2002)

coworkers (Wang et al., 2013), in which theyoligo-deoxynucleotides,

toinduceand RNA interference (RNAi) (McManus et al., 2002;

successfully used CRISPR–Cas9 in a one-step process to

Hannon et al., 2002) to suppress the expression of

produce mice with mutations in multiple genes. This

specific genes offered the possibility of producing

group

transgenic livestock more efficiently during 2002.

co-injecting fertilized oocytes at the pronuclear stage

CRISPR–Cas9 has revolutionized the generation ofwith not just the Cas9 mRNA and sgRNA, but alsotransgenic animals. This system has demonstrated ansingle-stranded DNA oligos, enabling HDR based on theunprecedented efficiency, multiplexability, and ease of

DNA oligos as templates. Blastocysts with these DNAuse, thereby reducing the time and cost required foroligo injections were implanted into the wombs of foster

genome editing and enabling the production of animals

mothers. The time scale required for the generation ofwith more extensive genetic modifications (Shrock et al.,mice carrying multiple mutations using pronuclear2017). Other strategies for increasing the efficiency of

injection was shown to be approximately one month,

CRISPR/Cas9 genome editing include inhibition ofmuch shorter than the six-month to year-long time scale

NHEJ (non-homologous end joining) or enhancement of

required by conventional methods. In addition to

HDR (homology directed repair) through chemical

germline editing, ex vivo and in vivo gene editing has

treatments (Maruyama et al., 2015; Nakao et al., 2016).

been

Methods

developed to perform in vivo CRISPR–Cas9 editing in

The CRISPR–Cas9 system has been used to generate arodents. The necessary reagents are generally packaged

wide variety of transgenic animals via a number ofin lentiviruses or AAV. Multiple tissues, such as lungdemonstrated

demonstrated.

preciseMultiplegenomesystems

editing

have

by

been

common methods. These include: (i) germline editing via(Sánchez-Rivera et al., 2014), neurons (Swiech et al.,injection or electroporation of CRISPR–Cas9 reagents2014), or liver (Cheng et al., 2014) have been targeted.

into early-stage zygotes or gonads, often used in model

Delivery strategies using cationic nanoparticles have alsoorganisms, such as Caenorhabtidis elegans, Danio rerio,

been developed (Zuris et al., 2014).

and Drosophila melanogaster, as well as in larger

Larger Animals The production of larger transgenic

animals, (ii) primary cell editing followed by somatic

mammals, including pigs (Li et al., 2015) and cows (Gaocell nuclear transfer (SCNT), often used in larger animals,

et al., 2017), has been accomplished via SCNT and/or

such as pigs and cows, and (iii) blastocyst injection withpronuclear

genetically modified ESCs followed by breeding toengineering primary cell lines in vitro, then fusing the

produce stable germline mutants, traditionally used innuclei of individual cells with enucleated porcine oocytes

mice in the form of plasmids, mRNA preparations, as

to produce genetically modified embryos (Verma et al.,RNPs, or in viral vectors such as AAVs (Shrock et al.,2015). At the blastocyst stage of development, these2017).

embryos are delivered to the uterus of a surrogate mother.

Small Model Organisms In the fruit fly Drosophila,

Transgenic pigs may also be generated using zygote

injection.

SCNT

involves

genetically

CRISPR–Cas9 can be efficiently used to generate

injection of the CRISPR–Cas9 reagents (Wang et al.,germline genetic modifications. CRISPR–Cas9 reagents2016).
21

Proceedings of International Symposium on Animal Production and Conservation for Sustainable Development 2018
SESSION 1 - Avian Production and Reproduction -: 01

Nonhuman Primates Although NHPs require larger

promoters to drive secretion of the transgene into milk or

facilities and longer time periods for experiments as

an alternative is the use of kidney or bladder specificcompared with mice, they nonetheless play an important

promoters that direct transgene expression to the urine,role in biomedical research (Chan, 2013) as they aree.g., Prolactin for enhancement of immunity and Protein

phylogenetically closer to humans. The CRISPR–Cas9

C for blood coagulation. Transgenic cattle harboring

system has been of great value for genome editing inintact unrearranged human Ig heavy- and Ȝ-light-chain

NHPs. Indeed, highly precise genetic modifications inloci were created. These ‘transchromosomic’ cattle weremonkeys had not been feasible before the advent ofshown to produce human Ig. Primate-to-human organ

CRISPR–Cas9, as the only technologies available fortransplantation ushered the age of xenotransplantation. It

gene editing were viral vectors (Chan et al., 2001; Niu et

was recognized early on that for physiological,

al., 2010; Sasaki et al., 2009). CRISPR–Cas9 has been

anatomical, ethical and supply reasons the pig was theused to precisely edit the genome of the Cynomolgus

best choice as a donor animal for vascularized organs.

monkey. Using zygote injection, researchers introducedFirst published transgenic pig-to-primate xenograft used

Cas9 mRNA and five gRNAs targeting the Nr0b1,

a novel transgenic delivery system for complement

PPar-gamma, and Rag1 genes (Niu et al., 2014). In theregulatory proteins in human (Byrne et al., 1997).

future, it is thought that genetically engineered NHPs

Transplant organs may soon come from transgenic

may be very useful to study neurological disorders andanimals. Currently, xenotransplantation is hindered by ahuman traits that cannot be easily recapitulated inpig protein that can cause donor rejection but research iscommon model organisms.

underway to remove the pig protein and replace it with aApplications

human protein.
obesityandTransgenic Livestock Dairy cattle are likely candidates

reproductive

andfor transgenesis if the mammary gland is to be used as ahaematological disorders, providing future hope for abioreactor, as they produce about 10,000 liters ofvariety of human therapeutic interventions through genemilk/year with 35 g protein/liter. The animals could betherapy. Transgenic animals can be used for disease

made to secrete nutraceuticals in milk that may have an

resistance in humans and animals. Clements et al.

impact over the growth of offspring. Physio-chemicalreported that transgenic sheep have been developed thatproperties of milk get altered while improving the milk

is resistant to Visna virus infection (Clements et al.,composition and casein variants are the main target.

1994).

spongiform

Brophy et al. reported that cloned transgenic cattle haveencephalopathy (Scrapie) is also prevented by the knockbeen developed that produce increased amounts of beta

down of prion protein (Weissmann et al., 2002).

and kappa casein in milk that increase the value of milk

Transgenic mice have been developed that secretes

in the production of milk-based products like cheese,recombinant antibodies in milk to neutralize the corona

yoghurt and also increase the shelf life of milk products

virus

gastroenteritis(Brophy et al., 2003). Transgenic animals could also be(TGEV), an economically important disease in case ofmade to secrete antibodies in their milk that givepigs (Castilla et al., 1998). Transgenic dairy cows

resistance against several diseases like mastitis or tosecreting lysostaphin into their milk have advanced

secrete antimicrobial peptides like lysozyme. Grosvenor

resistance to mastitis due to protection provided by

et al. reported that the milk composition could also belysostaphin. It kills the bacteria Staphylococcus aureus inchanged by making the transgenic animals to secretea dose dependent manner that protects the mammary

growth factors in milk consequently, affecting the growth

gland against this major mastitis-causing pathogen

and maturation of newborn offspring (Grosvenor et al.,(Donovan et al., 2005).

1993). Varieties of monoclonal and recombinantOncomouse was first transgenic animal to be patented.

antibodies were produced in transgenic goats and cattle

An activated human oncogene sequence is introduced(Houdebine et al., 1993; Grosse et al., 2004). Kuroiwa et

into the animal at an early embryonic stage and it isal. reported that transchromosomal animals could befound in its germ and somatic cells. The production ofused for the production of human therapeutic polyclonal

Biomedical

Research

immunological,

The

Modelsneurological,

transmission

responsible

for

of

for

bovinetransmissible

therapeutic proteins from transgenic animals usuallyantibodies (Kuroiwa et al., 2002).

involves their expression from mammary-gland specific

Transgenesis research with sheep, goat or pigs has

22Proceedings of International Symposium on Animal Production and Conservation for Sustainable Development 2018
SESSION 1 - Animal Production and Biotechnology -: 01

concentrated in the most part on utilizing their mammary

animals, studies of various disease models andglands as bioreactors for production of pharmaceuticalproduction of pharmaceuticals. However, there are many

proteins. Several conventional methods were used for thevital problems that need to be resolved for transgenic

production of therapeutic proteins using bacteria, plants,

animal studies.

yeast etc. but most of them were deficient in machinery

Dietary and Food Safety Concerns Food safety offor post translational modifications of eukaryotic genes.

bioengineered products is always a significant public

The transgenic livestock serve as potential bioreactors

topic. Some of the foreign gene and its promoter

for the production of valuable proteins. Proteins likesequences from the virus may occur in the recipient

antithrombin III (AT III), tissue plasminogen activator

animals(TPA) and antitrypsin have been derived from therecombination or integration may cause the formation of

mammary gland of transgenic sheep and goats. The

new virus. Foreign gene inserted in the chromosome

human AT III (for the treatment of heparin resistantlocus may also result in different genetic changes inpatients) is expected to be in market (Kues et al., 2004).

different degrees, causing unintended effects. Transgenic

Glycosidase has been produced in the milk of transgenic

animals may cause allergic reactions and there is

rabbits, which is used in the treatment of Pompe diseases

increased risk of zoonotic diseases.

(Van et al., 2001). Role of transgenic animals in carcass

Environmental Impacts If transgenic animals are in thecomposition and growth enhancement are introduction of

external environment and mating with wildlife thenchicken ski gene has caused muscular hypertrophy inforeign gene may spread, which results in changing thecase of pigs and cattle (Bowen et al., 1994). The acidspecies composition of the original genes, causing

meat gene or Rendement processing Napole gene has

confusion in species resources. Decline in the genetic

been involved in low yields of pork there by affecting thediversity may be there due to loss of wild allele. Once

quality of meat in pig. Silencing the expression of thisreleased into the environment, transgenic animals cangene in pigs improve the quality of meat and modify the

disturb the ecological balance of species and genetic

post mortem pH. There was a significant improvement indiversity of threatened species.

growth rate and feed conversion in transgenic pigs with

Ethicshuman metallothionein promoter (Nottle et al., 2001).

As recent technology has accelerated the ability to create

Transgenic swine has been developed for production oftransgenic animals, ethical discussions have become

functional hemoglobin having same oxygen binding

increasingly relevant. Debates have been most intense

capacity as that of normal human hemoglobin. It could

regarding editing of the human genome, especially of thebe purified from porcine blood (D’Agnilalo et al., 1998).

germline, but have also concerned editing of nonhuman

The main potential application of transgenic animal isanimal genomes. For example, the proposal to use

the production of recombinant and biologically active

CRISPR–Cas9 gene drives to eradicate alleles or entire

in

case

of

transgenesis.

Homologous

proteins in the mammary gland and this in turn could bespecies from ecosystems has been the subject of ethicalused for the benefit of mankind. This is known as “Geneconsideration. Use of animals in the transgenic research

Pharming”. Mammary gland is the favored site forleads to greater suffering to the animals. But most people

production of these proteins since large quantities can beseem to accept some animal suffering to serve the basic

extracted and purified (Houdebine et al., 1993). Farminterest and human welfare. Using animals for the

animals like cattle and pigs could be used as anproduction of therapeutic proteins, we reduce them toappropriate model for the study of human diseases likemere factories. Animals also are living beings and feel

cystic fibrosis, cancer and neuro-degenerative diseases

pleasure and pain just as we do. Animal sufferings are

and their therapies (Li et al., 2003). Pigs could be used asalso caused by the expression of transgenes, inducing

an effective model for the study of growth hormone

tumors or neurodegenerative diseases. Geneticallyreleasing hormone (GHRH) defects (Draghia et al.,altering the cells of an animal can lead to unknown side1999).

effects resulting from modifying genes. Even though

Limitations

humans may benefit from transgenic animals, the animal

Transgenic animals have large number of applications

itself may not be benefited. Foreign genes might affectforthe host and bring threats to ecological balance andtheimprovementofanimalproduction andspecies diversity (Miao, 2013).

reproduction quality, improvement of productivity in23

Proceedings of International Symposium on Animal Production and Conservation for Sustainable Development 2018
SESSION 1 - Avian Production and Reproduction -: 01

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Transgenic animal production techniques have developed
rapidly in the recent past and provided platforms for the
preparation of transgenic animals. These techniques
provide an entirely new alternative for the accurate
modulation of genes for human use, particularly the
production

of

drug,

organ

culture

for

human

transplantation and their use in biomedical research will
be significant. It is expected that with the development ofsimple and novel animal transgenic techniques will leadto more benefits and may help to alleviate or provide tocure certain diseases, which are presently incurable or
expensive to treat. Production of transgenic animals andrelated products is still infancy in Bangladesh.
Transgenic animal could be produced using different
method, but no method seems to be perfect and has its
own pros and cons. Despite these facts, transgenic
animal production has a great prospect in animal
pharming for human use.

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Proceedings of International Symposium on Animal Production and Conservation for Sustainable Development 2018
SESSION 1 - Animal Production and Biotechnology -: 02

miRNAs and Jenes interaction as potential biomarker for farm animal fertility
Sigit Prastowo1,*, Ahmed Amin2, Nuzul Widyas1 and Adi Rayriyanto1
12

Department of Animal Science, Sebelas Maret University, Surakarta, Indonesia
Department of Animal Production, Faculty of Agriculture, Cairo University, Egypt*
Corresponding author: prastowo@staff.uns.ac.id

SUMMARY
In current farmed animal practices, the success of a breeding program depends upon conventional animal reproduction.
It is closely related to both male and female fertility which indicates the ability of an animal to produce offspring.
Animals’ fertility is often disturbed causing infertility cases which lead to genetic and economic loss especially whenoccur in superior animal population. MicroRNAs (miRNAs) are small noncoding RNA which work posttranscriptionally to up or down regulate gene expression by binding into 3-UTR mRNA of specific target gene. One or
many miRNAs can interact with many genes or with only one gene as the target. The aforementioned interaction was
suggested as a fine tune process of gene expression which control specific function of animal metabolism in general, or
specifically in animal fertility. There were many genes which have been reported to be responsible for specific function
in animal reproduction process and these genes expressions were under the role of miRNAs. Hence, miRNAs and theirinteractions with genes which control male and/or female reproduction carry the potentials to be developed as fertility
biomarkers. However, in every interaction occurrence between miRNA and gene related to its specific functions, further
wet lab study is required as a validation procedure.
Keywords: miRNA, potential biomarker, farm animal fertility

miRNAs in animal reproduction both in male and female
fertility, and an insight pipeline of miRNAs study tovalidate its interaction with specific gene expression.

Introduction
Micro RNAs (miRNAs) are non-coding RNAs that
regulate gene expression post-transcriptionally [1]. It isbelongs to a class of small (19–24 nt) regulatory RNA
which able to control many cellular process bymodulating the precise amount of proteins in the cell[2,3]. miRNAs have that roles through the recognition ofsequences between miRNAs and their target genes [4,5].
Both cellular and extracellular miRNAs are found to be
involved in regulating mammalian gene expression [6,7].
Because of its broader targeting abilities, miRNAs are to
be found involved in most of biological processes andcellular pathways [8]. miRNAs are comprised of 1-5% ofmammalian genome and according to bioinformatics
analysis there are more than 60% mammalian genes
could potentially target by a single miRNA [9]. Recently,
38.589 mature miRNAs were annotated in miRBase(http://www.mirbase.org/ released 22 at March 2018). In
that database, there are 4719, 2145, 877, 4612, 260, and1414 mature sequences for bovine, chicken, porcine, goat,
sheep, and equine miRNAs respectively.
Similar to other biological process, most of animal
reproduction event also controlled by many genes
expressions. For example, miRNAs expression andregulation have been observed in bovine sperm [10],oocyte[11], ovary [3], zygote [12], and preimplantationembryo [13]. Previous study discover miRNAs
expression in tissue specific and have temporal
expression pattern in livestock [14]. However, to the bestof our knowledge there were little studies performed toobserve the role of miRNAs in animal reproduction bycontrolling
specific
gene
expression.
Those
aforementioned background bring the authors, to show
brief idea of using miRNAs as biomarker of farm animal
fertility which control gene function in reproduction. This
paper will cover information regarding miRNAbiogenesis and its role in gene expression, the glance ofA brief of miRNA biogenesis and its role ingene expression
miRNA biogenesis begins in the nucleus [15], initiatedby miRNA gene transcription with the help of RNApolymerase II providing long precursor transcripts know
as pri-miRNA. Mediated by Drosha and DGCR8A, primiRNA then proceeds into hairpin structure called premiRNA which has shorter sequence. The pre-miRNA
then transported to cytoplasm facilitated by exporting 5
as transport enzyme. The pre-miRNA then edited by
DICER resulting mature RNA, approximately 22 nt, andready to target specific mRNA.
In its action, mature miRNA and Ago protein form aribonucleoprotein complex, RISC, mediating posttranscriptional gene activity by bind to 3-UTR of mRNAtarget gene [15]. miRNA down regulate gene expression
through mRNA translation inhibition or degradation.
Several reports also reporting that miRNA can befunction as post transcriptionally stimulation of gene
expression [16–18]. Both down and/or up regulation ofgene expression are the process of cellular fine tune ororchestrated protein level [19].

miRNA in male farm animal reproduction
Considering the role of male as sperm source, in thispaper we only show information of miRNAs which havefunction or related to spermatogenesis and sperm quality
parameters. Several studies, listed in Table 1, shows
miRNAs related to spermatogenesis, sperm motility, andsperm abnormality. Sperm production and its quality are
the most considerable trait for male fertility [20]. Any
disturbance of sperm quality, would lead to low
conception rate [21] or egg hatchability [22]. In thisregards, losing fertility in male sometimes difficult to26

Proceedings of International Symposium on Animal Production and Conservation for Sustainable Development 2018
SESSION 1 - Animal Production and Biotechnology -: 02

diagnose and the major problems are coming fromgenetic defect [23].

to the oocytes development [28]. Both expressions werelower in oocytes, compared to cumulus and granulosa
cells. It is well known that cumulus cells have an
essential function in oocytes maturation [35], while after
ovulation granulosa cells turn into luteal cells to produce
progesterone for pregnancy support [36]. Other study
reporting miR-19b, miR-130, miR-101, and miR-19a
were predicted to be involve in bovine granulosa cellluteinization [29]. miR-27b, miR-371, and miR-382 werereported to be involve in preovulatory growth of equinefollicle by altering ID2 levels [30], while miR-34a playrole in granulosa cell apoptosis by targeting the INHBB
gene in porcine ovary.
In the reproduction event related to embryo, we can show
two studies of miR-212 and miR-196a were determining
embryo preimplantation in cattle. Both miRNAs are
expressed in oocytes and tends to increase in four andeight cell stage of embryos followed by a decline atmorula and blastocyst stages. miR-122 demonstrated a
temporary
regulation during maternal to zygotic
transition in bovine embryos by negatively regulate
FIGLA mRNA [32]. Meanwhile miR-196a has beenvalidated to be negative target of Newborn ovary
homeobox gene (NOBOX) mRNA [33] which isimportant for early embryonic development.

Table 1. miRNAs in male animal reproduction
miRNA namemiR-202
ssc-miR-10a-5p
ssc-miR-125b
ssc-let-7f
ssc-miR-186
let-7alet-7dlet-7emiR-22
miR-17-5p
miR-26a-5p
miR-486-5p
miR-122-5p
miR-184
miR-122-5p
miR-184
miR-20a-5p

Pathway

AnimalRelatedreproduction
process
Spermatogenesis
Spermatogenesis

Status

Source

-

[24]
[25]

-

Chicken
Porcine-PorcineSperm motility
and abnormality

-

[26]

PTEN

BovineHigh and low
sperm motility

-

[14]

PI3K/AKT
Signaling
STAT3

BovineHigh and low
sperm motility
High and low
sperm motility

-

[14]

-

[14]

BovinemiRNAs are play critical role in the regulation of target
genes during spermatogenesis [27], for that they haveclose relationship with male infertility. In the previous
study, miR-202 found to be upregulated in developing
male chicken gonad, and also can be indicator for sexual
dimorphic in chick embryo [24]. miRNA ssc-miR-10a-5p,
ssc-miR-125b, ssc-let-7f, and ssc-miR-186 were reported
highly expressed in porcine spermatogonia, pachytene
spermatocytes, round spermatids, and spermatozoa
respectively [25]. However, there were no information inboth studies, to which gene those miRNAs targeted.
In sperm motility, it is reported that miRNA let-7d andlet-7e were higher express in porcine sperm with low
motility. Furthermore miRNA let-7a, let-7d, let-7e, andmiR-22 were significantly increase in abnormal sperm
when compared to the normal one [26]. Again, that study
didn’t show any gene as the miRNA target [26]. While inbovine, previous report show miR-17-5p, miR-26a-5p,
miR-486-5p, miR-122-5p, miR-184, and miR-20a-5p
have different expression in high and low sperm motility[14]. According to that study, these miRNAs are targeting
PTEN and STAT3 pathway, and PI3K/AKT signaling.

miRNA validation study
In animal production area, growing evidences shows thatmiRNA regulate gene expression subsequent to animal
production traits. For instance, there is a different of
miRNAs expression between bulls with high and low
fertility [37]. miRNA also involved in milk production[38], control postpartum period [39], chicken growth andcarcass trait [40], pregnancy [41], and embryo viability[42]. Considering that promising functions, more study
needs to be performed to understand the miRNA role andits application to animal production as biomarker or as a
production enhancer agent [43].
So far, the bioinformatics prediction indicated thatmammalian miRNAs able to regulates 30% of all proteincoding genes [44]. Most of miRNA control gene
expression by binding to 3-UTR sequence of mRNAtarget gene at nucleotides number 2-8 which known as
seeding region or binding site [45–47]. Since there arenumbers of genes targeted by one miRNA or vice versa,
the validation study of each miRNA targeted specificgene need to be performed. This work would inform theexact function of miRNAs or genes in the animal
reproduction specifically.
As our experiences, miRNA validation study covering
steps in dry and wet lab. Dry lab consists of in silico
study for miRNA target prediction to specific gene,
miRNA sequence similarity analysis, prediction of
miRNA target in 3-UTR mRNA gene. Moreover, in wet
lab covering plasmid construction by 3-UTR clone,followed with cell culture, and luciferase assay analysis
which commonly performed using cell model.

miRNA in female reproduction
Several studies listed in Table 2, shows the function of
miRNAs in female animal reproduction. These, various
reproduction events are related to fertility in female
animal. In this review, we only show studies which wererelated to ovaries, oocytes, and embryos.
Table 2. miRNAs in female animal reproduction
miRNA
name
miR-106a
let-7
miR-19b
miR-130
miR-101
miR-19amiR-27bmiR-372
miR-382
miR-34amiR-212
miR-196a

Gene(s)AnimalRelated reproduction
process

Status

SourceMYC
WEE1A
AMPKA1

CattleOocytes

Predicted[28]

CattleGranulosa cells

Predicted

[29]

ACVR1
ID2

EquineFollicle growth andmaturation

Predicted

[30]

INHBB
FIGLA
NOBOX

PorcineCattleCattleGranulosa cell apoptosis
Preimplantation embryo
Preimplantation embryo

Validated
Validated
Validated

[31]
[32]
[33]

In silico study
targeted to gene

Oocytes known as female gamete which ovulated as theresult of ovarian activity [34]. Any disturbance of ovarian
activity lead to the female infertility as well as reducing
reproductive efficiency. It would also increase thepotential of economic loss in animal farming activity.
Previous report shows miR-106a and let-7i were related

toprediction

miRNAs

According to the previous study [29], there were threesteps as pipeline of in the miRNA prediction by in silico
study method. First, miRNAs prediction tolls (databases)
were employed to predict miRNAs targeted to specific27Proceedings of International Symposium on Animal Production and Conservation for Sustainable Development 2018
SESSION 1 - Animal Production and Biotechnology -: 02

gene, namely MirTarget2 (www.mirdb.org), PicTar(http://pictar.mdc-berlin.de/),TargetScan(www.targetscan.org)
andmiRanda(http://microRNA.org). Another database also possible tobe employed. Thereafter, common miRNAs that were
found in all these databases were selected as candidate.
Venn diagram would be great tool to select common
miRNA as mentioned in the previous study [48].

Luciferase assay in cell model
Activity of the luciferase gene in the constructed plasmid
can be measured using cell culture. In this regard cell
types could be varied. The cells seeded at 2 x 105/Pl in 24
wells plate and transfected with match and mismatch
pmirGLO luciferase vector at confluency >70%. Alongwith that miRNA mimic and inhibitor also transfectedusing Lipofectamine 2000 as transfection agent. After 24hour of culture, cell lysate then collected and store at 80°C until dual luciferase reporter assay performed [50].
Renilla activities is measured using a dual luciferase
assay protocol on a Luminometer. The level of luciferase
was compared to the un-transfected cells to ensure aproper transfection of the cells. The relative activity wascalculated by normalization with the firefly activity.

miRNA sequence similarity analysis
In the market, the availability of miRNAs primer iscommonly specific for human study, therefore for future
miRNAs profiling in farm animal, a similar primer
sequence to human will be use. For this, similarity
sequence analysis become a reasonable thinking. The
mature sequence of selected miRNAs candidate accessedfrom miRbase (www.mirbase.org), followed withsequence similarity analysis between human (hsa) vsanimal miRNA, for example bovine (bta), avian (gga),
porcine (ssc), goat (chi), sheep (oar), and equine (eca).
These three letters in the bracket are identification for
specific animal species. As an example, from previous
study, Table 3 show the similarity analysis between
human (hsa) vs bovine (bta) miRNAs.

Future perspective
A number of miRNAs expression have been identified
related to both male and female reproduction events. Its
expression, though not directly targeted to specific gene,already demonstrated as indicator of fertility in farmanimal reproduction. There still long way to know eachmiRNA role to specific gene lead to the reproduction
process. In this point, thousand interactions could be
revealed between miRNA, gene, and fertility.
To know the exact function of selected miRNAs
candidate, a series of profiling and validation study need
to be performed. Moreover, along with the improvementof computational algorithm features in miRNA target
prediction software, the more precise prediction for
miRNA candidate selection would be resulted. So far, thecomputational method is still the golden attempt to startin revealing miRNA and gene interaction or in contrary.

Table 3. miRNA sequence similarity analysisa
miRNA

miRNA name

Mature sequence (5’-3’)*hsa-miR-323a-3p
CACAUUACACGGUCGACCUCU
bta-miR-323
CACAUUACACGGUCGACCUCU
miR-23ahsa-miR-23a-5p
GGGGUUCCUGGGGAUGGGAUUU
bta-miR-23a
AUCACAUUGCCAGGGAUUUCCA
miR-221hsa-miR-221-5p
ACCUGGCAUACAAUGUAGAUUU
bta-miR-221
AGCUACAUUGUCUGCUGGGUUUa*)Source: [29,48]; www.miRbase.com

Similarity(%)

miR-323100-

Prediction of miRNA binding sites in 3-UTR
gene

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also cloned in pmirGLO vector.

28

Proceedings of International Symposium on Animal Production and Conservation for Sustainable Development 2018
SESSION 1 - Animal Production and Biotechnology -: 02

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Luteinization: An In Silico Study Proceeding of the
1st International Conference on Tropical
Agriculture ed Alim Isnansetyo and T R Nuringtyas(Yogyakarta: Springer International Publishing AG)
pp 431–8[30] da Silveira J C, Carnevale E M, Winger Q A and
Bouma G J 2014 Regulation of ACVR1 and ID2 bycell-secreted exosomes during follicle maturation in
the mare Reprod Biol Endocrinol 12 44[31] Tu F, Pan Z X, Yao Y, Liu H L, Liu S R, Xie Z and
Li Q F 2014 miR-34a targets the inhibin beta B gene,promoting granulosa cell apoptosis in the porcine
ovary Genet Mol Res 13 2504–12[32] Tripurani S K, Wee G, Lee K B, Smith G W, Wang
L and Jianboyao 2013 MicroRNA-212 posttranscriptionally regulates oocyte-specific basichelix-loop-helix transcription factor, factor in the
germline alpha (FIGLA), during bovine early
embryogenesis PLoS One 8 e76114[33] Tripurani S K, Lee K B, Wee G, Smith G W and
Yao J 2011 MicroRNA-196a regulates bovine
newborn ovary homeobox gene (NOBOX)

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Proceedings of International Symposium on Animal Production and Conservation for Sustainable Development 2018
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expression during early embryogenesis BMC Dev
Biol 11 25[34] Senger P 2005 Pathway to Pregnancy andParturition (Washington: Pullman)[35] Tanghe S, Van Soom A, Nauwynck H, Coryn M
and De Kruif A 2002 Minireview: Functions of the
cumulus oophorus during oocyte maturation,
ovulation, and fertilization Molecular Reproduction
and Development 61 414–24[36] Murphy B D 2000 Models of luteinization Biol
Reprod 63 2–11
[37] Govindaraju A, Uzun A, Robertson L S, Atli M O,
Kaya A, Topper E, Crate E A, Padbury J, Perkins A
and Memili E 2012 Dynamics of microRNAs in bull
spermatozoa Reprod Biol Endocrinol 10 82[38] Jabed A, Wagner S, McCracken J, Wells D N and
Laible G 2012 Targeted microRNA expression indairy cattle directs production of beta-lactoglobulinfree, high-casein milk Proc Natl Acad Sci U S A 109
16811–6[39] Fatima A, Lynn D J, O’Boyle P, Seoighe C andMorris D 2014 The miRNAome of the postpartum
dairy cow liver in negative energy balance BMC
Genomics 15 279[40] Shi J and Sun G 2017 Effect of pre-miRNA-1658
gene polymorphism on chicken growth and carcass
traits Asian-Australasian Journal of Animal
Sciences 30 455–61[41] Ioannidis J and Donadeu F X 2017 Changes incirculating microRNA levels can be identified as
early as day 8 of pregnancy in cattle PLoS ONE 12[42] Pohler K G, Green J A, Moley L A, Gunewardena S,
Hung W T, Payton R R, Hong X, Christenson L K,
Geary T W and Smith M F 2017 Circulating
microRNA as candidates for early embryonicviability in cattle Molecular Reproduction andDevelopment 84 731–43[43] McDaneld T G 2009 MicroRNA: mechanism ofgene regulation and application to livestock J AnimSci 87 E21-8[44] Filipowicz W, Bhattacharyya S N and Sonenberg N
2008 Mechanisms of post-transcriptional regulation
by microRNAs: are the answers in sight? Nat Rev
Genet 9 102–14[45] Ambros V 2004 The functions of animal
microRNAs Nature 431 350–5[46] Bartel D P 2009 MicroRNAs: target recognition andregulatory functions Cell 136 215–33[47] Zhou P, Xu W, Peng X, Luo Z, Xing Q, Chen X,
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Jiang S 2013 Large-scale screens of miRNA-mRNA
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M H 2018 In-silico identification of microRNAs
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Prediction Current Genomics 10 478–92[50] Prastowo S 2015 Function and regulation of AMPactivated protein kinase in in vitro development andlipid metabolism of preimplantation bovine embryos(Rheinischen Friedrich-Wilhelms-Universität Bonn)

30

Proceedings of International Symposium on Animal Production and Conservation for Sustainable Development 2018
SESSION 1 - Animal Production and Biotechnology -: 03

Generation of functional oocytes of common marmoset
by xeno-transplantation of ovarian tissue
Shiori Miura1, Yoshitaka Maeda1, Jun Miyamoto1, Ena Nakatsukasa2,
Nobuyoshi Fujisawa1, Miki Miwa3, Katsuki Nakamura3, Kenji Sakimura2
and Toshikuni Sasaoka1,2
1.Department of Comparative & Experimental Medicine, Center for Bioresource-based Researches/Bioresource
Science Branch, Brain Research Institute, Niigata University
2. Department of Animal Model Development, Center for Bioresource-based Researches/Bioresource Science Branch,
Brain Research Institute, Niigata University
3. Primate Research Institute, Kyoto University

SUMMARY
Common marmoset (Callithrix jacchus) is well known being a non-human primate experimental animal which is usefulfor translational research, for instance, researches of human brain function and various human disorders. However, it isextremely challenging to deal with numerous primates and breed them. Therefore, a few research facilities can maintaina relatively large number of common marmosets. Only limited laboratories can certainly create a genetically modified
marmoset. Furthermore, considering that the reproduction engineering utilizing marmoset oocytes is not wellestablished, it is essential to develop the way to get lots of oocytes. In order to the efficient utilization of the ovary, we
have conducted xenotransplantation of the euthanatized marmoset ovaries to immunodeficient mice. Here we report thatfully developed oocytes of common marmosets have been obtained from xenografted ovaries of common marmosets ofimmunodeficient mice. These results demonstrate that ovaries of euthanatized marmoset can be used as a source ofgeneration of embryos. Now, we are studying at the effects of exogenous factors, i.e. reproductive hormone in order tocollect oocytes more efficiently from the fresh and cryopreserved ovaries.

Introduction

invaluable genetic resource, it can contribute not only to

The common marmoset (Callithrix jacchus) is a valuable

reducing the number of marmosets used but also tolaboratory animal, because of (1) similarity of metabolic,reducing costs.

physiological, and anatomical characteristics to those ofOvarian transplantation is one of the clinicalhumans, (2) ease and safety in handling due to its small

treatments to maintain an ovarian function. Thissize and (3) high reproductive efficiency. In recent years,

technique allows for retention of hundreds of immature

marmosets model is remarkable experimental model inoocytes kept within the original ovarian tissue. In thefields such as brain research, infectious disease,ovary of the marmoset, there are a lot of immature

reproduction,
superovulation

and
and

aging1–3.
in

The

vitro

methodsofoogonia than human 4. By effective utilization of

maturationofimmature oogonia existing in the ovary, we can obtain apreimplantation embryos have not been established.

sufficient

There are many difficulties in terms of breeding andproduction.

numberoffertilized

eggs

for

animalHere we report on the successful xenotransplantation

supplying of marmosets at a large scale.

of marmoset ovarian tissue to immunodeficient mice as

Therefore, we focused on the ovaries of marmosets

shown by the development of mature antral follicles.

who were euthanized in other laboratories due to the endof the experiment or poor physical condition. If we canobtain matured oocytes, these will be effective source forMaterial and Method

supplying oocytes even in a small-scale laboratory. By

Ovaries were taken from euthanized adult marmosets

reusing a tissue that is originally disposed as an

and transported in Lifor solution at 4°C (Fig 1A). The

31

Proceedings of International Symposium on Animal Production and Conservation for Sustainable Development 2018
SESSION 1 - Animal Production and Biotechnology -: 03

tissues were dissected into approximately 1 to 2 mm3

Following xenotransplantation, it is confirmed the(Fig 1B) using a scalpel and scissors in Lifor solution

development of follicles at 600 ȝm or more in theand placed on ice until transplantation.

recipient mouse with stimulation of rhFSH. HistologicalOvarectomies of recipient mice were carried out priorevaluation showed the existence of normal eggs in theto grafting to eliminate the influence of mouse ovaries

follicle morphologically (Fig 3).

(Fig 1C). Transplant surgery was performed on the left
and right ovaries and kidneys as follows. The kidney was
taken out of the body. The kidney capsule was incisedwith micro tweezers or injection needle, and a rounded
glass tube was inserted to make a pocket. One to three
Fig 3. (A) Follicular development in grafts ofmarmoset ovarian tissue after xenotransplantation.
Antral follicle in graft of tissue recovered 21days
rhFSH treatment. Scale bar=1000m, and oocyte(B) Scale bar=300m.

ovary pieces were placed in pocket and the kidneys werereturned to the abdominal cavity (Fig 1D).

Throughout the experiment period, it was revealedthepresence of morphologically normal

oocytessurrounded by cumulus cells. Depending on rhFSH
stimulation, the number of oocyte tends to increase (table1). In our experiments, it is difficult to collect samples
with adjusted estrous cycle of recipient mice because weuse ovaries took from the animals euthanized due to thecompletion of experiment or healthy condition. In

Fig.1 Ovary transplantation. The marmoset ovary (A)was dissected into approximately 1 to 2 mm3 (B).
Ovarectomies were carried out prior to grafting (C),and ovary pieces were implanted under the kidney
capsule (D).

marmoset, administration of FSH on day 9 or 11
improves the quality of eggs and fertilization rate of invitro fertilization rate significantly5. More detailed
examination is required as to whether this condition issimilar to the case where the graft ovaries are

From 18th day after marmoset ovarian transplantation,
animals were administered with 7.5 units / 0.1 mL oftransplanted into nude mice.

recombinanthumanfolliclestimulating

hormoneTable 1. Frequency of rhFSH stimulation and number(rhFSH) every 24 hours subcutaneously. The graft tissuesof oocyte in the graft follicles.

were stained with hematoxylin and eosin solution forDays after stimulation

histological evaluation of ovaries.

of rhFSH

Number of oocyte




0

1



7

2



In this experiment, we aimed to obtain fertile oocytes by

14

3



xenotransplantation of euthanized marmoset ovary to

21

1



immunodeficient mice. The graft tissues survived under

30

4



Result and Discussions

the kidney capsule for 31 days after transplantation (FigNow, we are trying to establish experimental

2).

procedures regarding the hormone administration at
pre/post transplantation, the storage method or freezing
method of the ovary and grafting conditions to improveovarian function after transplantation. We are aim toestablish an economical and reliable systems enabling
generalization of genetically modified marmoset modelby ovarian xenotransplantation.
Fig 2. Engraftment of transplanted tissue.

32Proceedings of International Symposium on Animal Production and Conservation for Sustainable Development 2018
SESSION 1 - Animal Production and Biotechnology -: 03

Conclusion
In this study, we succeed in the euthanasia marmoset
ovary xenotransplantation to a nude mouse and it ishistological confirms the survival of the grafts ovary
morphologically.

Acknowledgement

We gratefully acknowledge to Drs. Erika Sasaki, Yoko
Kurosaki, Takafumi Inoue of Central Institute forExperimental Animals to providing
many advices.

Reference
1. Mansfield K. Marmoset models commonly used inbiomedical research. Comp Med. 2003;53(4):383-392.
2. Okano H, Hikishima K, Iriki A, Sasaki E. The
common marmoset as a novel animal model system forbiomedical and neuroscience research applications.
Semin Fetal Neonatal Med. 2012;17(6):336-340.
3. Mitchell JF, Leopold DA. The marmoset monkey asa model for visual neuroscience. Neurosci Res.
2015;93:20-46.
4. Fereydouni B, Drummer C, Aeckerle N, Schlatt S,
Behr R. The neonatal marmoset monkey ovary is very
primitive exhibiting many oogonia. Reproduction.
2014;148(2):237-247.
5. Yoshioka M, Nobukiyo A, Hatakeyama T, et al.
Ovarian stimulation and in vitro maturation in thecommon marmoset. J Reprod Dev Suppl. 2010;56 (Suppl
Japanese Issue):J122.

33

Proceedings of International Symposium on Animal Production and Conservation for Sustainable Development 2018
SESSION 1 - Animal Production and Biotechnology -: 04

Marinobufagenin and preeclampsia in the squirrel monkey
Mohammad Nasir Uddin1,2,3, Syeda H. Afroze1,2,3, David CC Sprague2,3 and Thomas J Kuehl41

Texas A&M University College of Medicine, 2Orion Institute for Translational Medicine, 3Emergent Biotechnologies,
4

Baylor Scott & White Healthcare, Temple, Texas

SUMMARY
Our team seeks to understand the pathogenesis of preeclampsia (PreE), and develop strategies to lessen adverse
outcomes. This study focuses on marinobufagenin (MBG) as a trigger of preE in a nonhuman primate, the squirrel
monkey (Saimiri sciureus). We showed MBG in preE patients exceeds that in those with normal pregnancy. Others
showed angiogenic imbalance in patients developing preE. In a rat model of preE, we found MBG increases prior toangiogenic imbalance and development of preE. MBG administration to normal pregnant rats induces angiogenic factor
alterations, hypertension, proteinuria, and intrauterine growth retardation. We propose the novel hypothesis that MBG
produces preE in a nonhuman primate and that this effect can be blocked or attenuated by RBG, and or a MBG
antagonist. We proposed two hypothesizes: 1: We demonstrated that administration of MBG in early pregnancy leads toangiogenic factor imbalance and the onset of preE symptoms and signs in otherwise normal squirrel monkey
pregnancies. The working hypothesis is that MBG elaboration early in the pregnancy induces hypertension, proteinuria,
and angiogenic factor imbalance (increasing the angiogenic factor ratio) during and potentially following in vivo
exposures. 2: We compared the ability of RBG and anti-MBG monoclonal antibody to attenuate the effects of MBG invivo, using the squirrel monkey model. We demonstrated that MBG triggered preE features in pregnant squirrel
monkeys including maternal effects and fetal growth retardation, which can be blocked by treatment with RBG, astructurally similar antagonist, or with anti-MBG antibody. These studies provide the first demonstration in a primate
species of the potential for blockade of MBG-triggered pathways.

Introduction and Significance
Preeclampsia (PreE) is a disorder of pregnancy occurringin 3-10% of gestations (Pridjian and Puschett, 2002;
WHO, 2000). The diagnosis of preE includes the de novo
onset of hypertension and proteinuria mid-gestation orlater, which may be accompanied by significant edema,
intrauterine growth restriction (IUGR), and CNS
complications. It is the second most frequent cause ofmaternal and fetal morbidity and mortality, accounting
for about 60,000 maternal deaths annually worldwide.
Marinobufagenin (MBG) is an endogenous mammaliancardiotonic

bufodienolide

withvasoconstrictive

properties (Bagrov and Fedorova, 1998; Fedorova et al.,
2001; Schoner, 2002). Increased circulating levels of
MBG

are

found

in

volumeexpansion-mediatedhypertension and in preE (Gonick et al., 1998; Lopatin et

MBG in the pathogenesis of preE in a primate and toal., 1999; Vu et al., 2005). In an animal model of preE

identify the therapeutic target. The objective of this(Ianosi-Irimie et al., 2005), the urinary excretion of MBG

proposal is to determine if MBG can cause features ofis elevated prior to development of hypertension andpreE and/or trigger the preE syndrome in a nonhumanproteinuria. Our overall goal is to understand the role ofprimate. If so, we will determine if the effect of MBG is34

Proceedings of International Symposium on Animal Production and Conservation for Sustainable Development 2018
SESSION 1 - Animal Production and Biotechnology -: 04

prevented by concurrent treatment with resibufogeninthat a vascular leak syndrome may develop in preE

(RBG), a structural antagonist of MBG, or with an(Chesley, 1972; Gallery et al., 1979; Hays et al., 1985). A

anti-MBG monoclonal antibody. (Fig. 1) is that MBG

syndrome with many of the phenotypic characteristics oftargets vascular beds and causes vascular responses thathuman preE results when pregnant rats are given weekly

induce the preE syndrome. This hypothesis is based on

injections of desoxycorticosterone acetate (DOCA), anddata in a rat model from our laboratory showing that

their tap water is replaced with normal saline

MBG elaboration occurs early in preE, which then(Ianosi-Irimie et al., 2005). This syndrome can be largely

causes the preE syndrome with hypertension, oxidative

reproduced by the daily injection of MBG, beginning instress, and angiogenic factor imbalance leading toearly pregnancy (Ianosi-Irimie et al., 2005). We assessed

endothelial dysfunction. The vascular response includes

vascular leak in this model and demonstrated greater leakalterations in the circulating balance of angiogenic

in preeclamptic rats (PDS) than in normal pregnant (NP)factors: both a decrease in the pro-angiogenic factors

animals (Uddin et al., 2009a). Angiogenic imbalance(VEGF,

theplays a role in the pathogenesis of preE in this rat model.

anti-angiogenic factors (sFlt-1, sEng). Administration of

However, the earliest event in the pathogenesis appears

MBG to rats beginning early in otherwise normal

to be the secretion and elaboration of MBG (Agunannae

gestations induces hypertension, proteinuria, and fetaleteffects, which are characteristic of preE. We are well

hyperpermeability of the endothelial cell layers byequipped to pursue this project because members of ouractivating the MAPK and apoptotic signaling which wasteam have developed a rat model of preE in which MBG

prevented by p38 inhibition (Uddin et al., 2009b). MBG

is elevated prior to the onset of preE features. We havealso disrupted endothelial cell junctions, an effectexperience with telemetry monitoring of blood pressure

prevented by p38 inhibitors (Uddin et al., 2009b). We

and hypertension that can be applied to the squirrel

found that the MAPK system is involved in themonkey. We have extensive experience monitoring

deleterious effects of MBG on cytotrophoblasts (CTBs),

pregnancy and fetal growth in small, easily handledimportant for normal placental development (Uddin et al.,nonhuman primates, including the squirrel monkey. The

2008a; 2008b). When resibufogenin (RBG), an MBG

rationale for the present study is based on our previous

antagonist, was given to rats that had been rendereddiscovery that MBG administration causes a preE

hypertensive and proteinuric in the manner described

PlGF,

TGF)

and

an

increaseinal.,2010).

In

these

studies,MBG

causedsyndrome in rats. The potential significance of theabove (Ianosi-Irimie et al., 2005), it reduced blood

current project is that successful completion will give uspressure to normal (Vu et al., 2006). The same was seena non-human primate model of preE and identify

in normal pregnant animals given MBG to induce

therapeutic targets that might effectively prevent thehypertension (Vu et al., 2005). When RBG was given

MBG-induced

patient

early in pregnancy, it completely prevented all of the PE

populations. A better understanding of these pathways inmanifestations noted in the rat model, including IUGRa primate has great potential for translation into clinicaland oxidative stress (Horvat at al., 2009, Uddin et al.

treatment and potentially prevention of preE.

2010a; 2010b; 2011). These data suggest that RBG or

preE

syndromeincertainSignificant, reliable, and reproducible data haveanti-MBG monoclonal antibodies (Fedorova et al., 2008)

accumulated which favor a view that preE is a disorder

may be important new therapeutic or preventativeof endothelial dysfunction (Roberts et al., 1989)

strategies for this syndrome. However, a primate model

involving oxidative stress. Methods for early diagnosis

to test this concept is not available. Preliminary studies

and treatment are unavailable, and prevention isindicate that MBG induces features of the preE

currently not possible. Therefore, this illness represents

syndrome

an important example of a medical unmet need. It seems

dysfunction, angiogenic imbalance, and intrauterine

clear that preE is not a single disorder, but a syndrome

growth retardation (IUGR). The major objective of this

with

and

proposal is to develop a primate model of preE andmechanisms (Pridjian and Puschett, 2002). Recent data

identify therapeutic targets that could effectively prevent

suggest that a significant segment of preE patients is

the MBG-induced preE syndrome.

responding to excessive secretion of marinobufagenin

Our team seeks to understand the pathogenesis of preE,(MBG) (Puschett, 2006). Obstetricians have suspecteddevelop novel diagnostic biomarkers to predict diseasemultiplepathophysiologicaltriggers

35

inpregnant

rats

includingendothelial

Proceedings of International Symposium on Animal Production and Conservation for Sustainable Development 2018
SESSION 1 - Animal Production and Biotechnology -: 04

severity, and develop new strategies to lessen adverse

demonstrate that administration of MBG in earlyoutcomes. There are no compelling nonhuman primate

pregnancy leads to angiogenic factor imbalance and themodels of preE, although preE features have been

onset of preE symptoms and signs in otherwise normal

reported in nonhuman primates (Palmer et al., 1979;

squirrel monkey pregnancies. Administration of MBG in

Hennessy et al., 1997; Podjarny et al., 2004), andearly or mid-gestation will lead to angiogenic factor

nonhuman primates share some features of implantation

imbalance and the onset of preE features in squirrelthat are important for understanding vascular dysfunctionmonkeys including hypertension, proteinuria, and fetal

related to eclampsia and fetal growth restriction (Wulff et

growth retardation. This hypothesis is supported byal., 2003; Carter, 2007). This application has a novel

publications cited above (A. Significance) and by the

focus on MBG as a potential trigger of preE features in aobservation that MBG is increased, and angiogenic

nonhuman primate, the squirrel monkey. We showed
MBG in preE patients exceeds that of those with normal
pregnancy (Abi-Ghanem et al., 2011). We and others
have demonstrated angiogenic imbalance in patientsdeveloping PE (Buhimschi et al., 2005; Molvarec et al.,2010; Abi-Ghanem et al., 2011). In a rat model of preE,
we found MBG increases prior to angiogenic imbalance
and that MBG acts on cultured cells to trigger
hyperpermeability

in

human

brain

microvascularendothelial cell monolayers through disruption of tightjunction proteins using MAPK pathways and to releasean angiogenic factor, sFlt-1, into the culture medium(Uddin et al., 2009a). MBG administration to normal

imbalance is observed in preE patients. Urinary MBG

pregnant rats induces angiogenic factor alterations,
hypertension,

proteinuria,

andintrauterine

and angiogenic factors were assayed in 30 preE and 40

growthnormal pregnant patients. The MBG levels were higher

retardation (Ianosi-Irimie et al., 2005; Agunanne et al.,in preE patients (Fig. 2; Abi-Ghanem et al., 2011).

2009). We now propose the novel hypothesis that MBG

Angiogenic imbalance was observed in preE patients

induces features of preE and potentially triggers preE incompared to normal pregnancies (Fig. 3). Our team is

a nonhuman primate and that these effects can beexperienced working with primate and the urinary levels

blocked or attenuated by RBG and or a MBG antagonist.

of MBG were measure in eight Owl monkeys at different

We propose to instrument and monitor blood pressure ingestational ages. The levels increase in early pregnancy

pregnancy, which is novel for this species, and to follow(~52 days) then decrease minimum (~80 days), thenother preE features including fetal growth effects using

increase toward term (~133 days) (Fig. 4).

minimally invasive techniques. If the results of thesetrials support the role of MBG as a cause of preE features
and that one or more agents can block this effect without
producing damaging fetal effects, it will open the door
for studies of a number of innovative treatments inprimates. Also, these studies may provide the first
demonstration in a primate species of the potential forblockage of MBG-triggered pathways.

Methodology, Results and Discussion
We propose the novel hypothesis that MBG produces
features of preE in a nonhuman primate and that thiseffect can be blocked or attenuated by RBG, an MBG
antagonist, or by anti-MBG monoclonal antibodies. We

36

Proceedings of International Symposium on Animal Production and Conservation for Sustainable Development 2018
SESSION 1 - Animal Production and Biotechnology -: 04

In this in vivo study, during one breeding season, 8

the approximately 20-week squirrel monkey gestation:

female squirrel monkeys will be identified in early

one in the first trimester (with treatment in weeks 4-7)

pregnancy with gestational sacs, prior to appearance ofand one in the second trimester (with treatment in weeks

the fetal pole with beating heart. They will be

11-14).
MBG is obtained from Cayman Chemical (AnnArbor, MI), with a purity of >98%. Identity and purity
will be confirmed by HPLC with MS analysis.
Beginning 1 week after instrumentation, a blood sample
will be drawn, and the animals (N=3 per group) will betreated with a minipump delivering 1) 7.65 ȝg/kg bodyweight / day of MBG in a DMSO/saline vehicle, or 2)
vehicle alone for 28 days. For 1 week prior to and for
four weeks of treatment, blood pressure and heart rate
will be recorded using telemetry twice each week. Urine
will be collected during the recording sessions (Table 1).
Blood will be collected weekly during this study interval
through the time of minipump removal and one week

instrumented with a telemetry device to report blood

after pump removal. Fetal growth and development willpressure and heart rate while freely moving during a

be measured by ultrasound weekly. Following thetelemetry recording session in a cage with urine

removal of the minipump, observations will continue forcollection capabilities for 3 hours once or twice per week.

the remainder of pregnancy with telemetry monitoring

We will assess the effect of MBG during two intervals insessions and urine collections once in every two weekand ultrasound assessment of fetal growth at two-week37

Proceedings of International Symposium on Animal Production and Conservation for Sustainable Development 2018
SESSION 1 - Animal Production and Biotechnology -: 04

monkey model. Our hypothesis is supported by materials

intervals.
Animals (N=3/group) treated during the second

cited above (A. Significance) and by the observation thatinterval will be instrumented with telemetry at the same

RBG corrects the BP in PDS and MBG-infusedtime as the first interval group, and their telemetry andhypertensive rats. When RBG was given early inurinary measures will serve as an additional control for

pregnancy, it completely prevented all of manifestations

manipulations. Similarly, the control group for the firstof preeclampsia noted in our rat model, including IUGRinterval can serve as an additional control for the treated

and oxidative stress (Fig. 5: Horvat at al., 2009, Uddin etanimals in the second interval. During each interval,al. 2010a; 2010b; 2011). When given to pregnant rats

three animals will be treated with vehicle, and three willrendered hypertensive by the administration of MBG,

be treated with MBG. Plasma from blood samples will

RBG resolves the hypertension (Fig. 6). Preliminarybestudies with a preE rat model: Female Sprague-Dawley

assayedfor

MBGlevels,angiogenic

andanti-angiogenic factors (VEGF, PIGF, TGF-B, sFlt-1,
and sEng). Urine will be assayed for protein levels,
creatinine, MBG, and angiogenic factors.
We anticipate that MBG will be increased in urine
and blood during MBG treatment and that blood pressure
will be increased. Proteinuria and fetal growth effects
may not be altered by these short-term exposures.
However, if MBG acts as a trigger, and provided 4 weeks
of treatment is sufficient to create vascular injury, thenwe expect that effects will continue, leading to aworsening of preE features as pregnancy progresses. We
anticipate that early treatment will be more effective thanlate treatment for this reason. Table 1 provides theschedule for animal manipulations in this experiment.
The effect sizes for blood pressure, MBG levels, andangiogenic factors are expected to be large based on
experience with pregnant rats, so that repeated measuresof a small number of pregnancies will likely be sufficient
to detect differences. Data will be expressed as means
with SE, and results will be analyzed using analysis ofvariance with repeated measures. Nine pregnant squirrel
monkeys will be used in this study during one breedingrats were mated with same-strain male rats, andseason. A pitfall of this study is that MBG at the dose

pregnancy confirmed by the presence of vaginal plug.

extrapolated from previous rat experiments may not bePregnant rats were divided into 4 groups: group I =sufficient to produce significant preE features in thenormal pregnant rats (NP); group II = pregnant rats thatsquirrel monkey during or after the 4-week exposure or

received deoxycorticosterone acetate (DOCA; 12.5 mgthat MBG may not trigger a worsening condition. If thiswas given in a depot form (i.p.) in the beginning of theis the case, a power analysis using data from the initial

experiment, followed by a weekly injection of 6.5 mg),experiment can be used to focus on a greater dose or

and whose drinking water had been replaced with 0.9%

longer exposure depending on how the blood levels of

saline (PDS, ‘pre-eclamptic’ = pregnancy + DOCA +MBG compare to those measured in patients with preE.

saline); group III = normal pregnant rats given daily

If effects are more prominent at one exposure time

injections of MBG (7.65 g/ kg/animal weight/day) oncecompared to another, this time will be used in a second

pregnancy was established on day four of the experimentexperiment during a second breeding season with

(NPM= normal pregnant + MBG). The group IV=PDS

another group of nine pregnancies.

rats were injected with anti-MBG-P antibody, 2.2

Compare the ability of RBG and anti-MBG monoclonal

mg/kg/d; on the 16th, 17th, or 18th day of pregnancy;

antibody to attenuate the effects of MBG in the squirrel

PDS-MAB= PDS + anti-MBG antibody-P (3E9), n = 10.

38

Proceedings of International Symposium on Animal Production and Conservation for Sustainable Development 2018
SESSION 1 - Animal Production and Biotechnology -: 04

Blood pressure was measured by the tail-cuff method

described by Watson, et. al. (2007) for the rat will be(IITC Inc., LifeScience Instruments, Model 59). At day

used in the squirrel monkey. Briefly, animals (about 800

19 of pregnancy, 24h urine collection was obtained.

g) are anesthetized with ketamine-xylazine. Then, aAnimals were euthanized on day 19, and pups weretransmitter is implanted (s.c.) in the back. An attachedcounted, weighed, and examined. The group parameterspressure catheter is placed in the aortic lumen via theare shown in Table 2. Vu et al. administered anright carotid. Animals are allowed to recover fromintravenous dose of MBG antibody (antiserum MBG-P,

surgery for one week before treatments are initiated.

Ab, 2.2 mg/kg) in PDS rats on the 16th, 17th, or 18th day

Telemetric baseline blood pressure and heart rate areof pregnancy (Vu et al., 2005; 2006).

recorded during 3-hour sessions in a single cage twice

If a preE syndrome is triggered, we will use the optimaleach week. Measurement of the following six parameters
occurs every 1 min. using the DataQuest System (Data
Sciences): systolic blood pressure, diastolic bloodpressure, mean blood pressure, heart rate, and activity
level. These data are maintained in computer files as foreach session for each animal (Fig. 7).

interval for MBG treatment in a second series that
divides MBG-treated pregnancies into three groups(N=3/group): 1) treated with MBG as in the firstexperiment, 2) given RBG at 30 ȝg/kg body weight / dayin a set of daily injections beginning prior to

Measurement of MBG:

implantation of the minipump delivering MBG, and 3)

MBG are measured by competitive ELISA according togiven anti-MBG antibody at 1 mg/kg body weight / day

the method most recently described by Abi-Ghanem et al.

in daily injections as for RBG. With data from Specific(2011). Urine samples are analyzed unprocessed, except

Aim 1, it will be possible to perform a power analysis tofor dilution with StartingBlockTM buffer. In contrast,

better predict the needed sample size and to estimate theserum samples are purified by solid phase extraction

effects of these manipulations on pregnancy losses. If a(SPE) on C18 Empore Disk Cartridges (Varian, Palo Alto,

progressing preE syndrome is not identified using shortCA). The disk is first conditioned with 150 L of

exposures, but short-term exposure is sufficient to induce

acetonitrile, centrifuged at 280 x g for 1 min., thenpreE features such as hypertension, the potential of RBG

washed with water and centrifuged as above. The serum

to block these effects can be measured. The treatment

sample (300 L) is then added to the disk with an equalintervals and sampling schedule for this specific aim will

volume of 10% acetonitrile in water, and centrifuged as

be similar to that used in the first experiment (Table 1),

above. Interfering substances are removed by sequential

and again the entire pregnancy will be evaluated towashes with Milli-Q water and 10% acetonitrile, and theoutcome variables.

sample is twice eluted with 150 L of acetonitrile.

Use of telemetry to measure blood pressure and heart

Samples are vacuum-dried and reconstituted in 100 Lrate throughout pregnancy. In each pregnant female, aof StartingBlockTM buffer. Diluted urine samples andradio frequency device will be implanted for independent

extracted serum samples are run in duplicate, andmonitoring of blood pressure and heart rate. The devices

average MBG concentrations (pg/mL) are extrapolated

from Data Sciences International (St. Paul, MN) as

from the standard curve using the StatLIA software.

39

Blood and urinary levels ofProceedings of International Symposium on Animal Production and Conservation for Sustainable Development 2018
SESSION 1 - Animal Production and Biotechnology -: 04

Measurement of angiogenic factors: The angiogenic
factors VEGF, PIGF, TGF-B, sFlt-1, and sEng aremeasured in the plasma and urine of the animals using
commercially available kits (R&D Systems, Minneapolis,and Assay Designs, Ann Arbor, MI) at various time
points of gestation (Agunanne et al., 2010).
Measurement of protein, creatinine, and other agents:
Urine protein excretion is measured using the pyrogallol
red method (Total Protein Kit, Micro Pyrogallol Red
Method, Sigma). Each animal is housed separately in a
metabolic cage. Creatinine is measured in blood andurine on a Nova 16 Analyzer (Waltham, MA), and thesquirrel monkey breeding colony. The infants born in thiscreatinine clearance (CCR) is calculated. Hematocrit isstudy will be measured and evaluated. They will be mademeasured using a StatSpin MP Multipurpose Centrifuge

available for other research uses. C-section for clinical(Norwood, MA) (Uddin et al, 2009a).

purposes is utilized in the squirrel monkey and

Selection of females, randomization, and baseline

subsequent pregnancy and delivery has been successful.

evaluations. Up to 9 pregnant adult females enrolledAsduring each breeding season (December through April ofeach year). Females will be randomly assigned tonormalanatomyandobtaindeliverlarge

infants,

animals will be cared for as appropriate by the animal

will be weighed and evaluated with real-time ultrasoundverifymonkeysneed for clinical use of surgical delivery. If this occurs,

treatment subgroups using a sealed envelope system. Alltosquirrelmalpresentation and maternal exhaustion can result in thefacility veterinarians. As animals may develop morebaseline

severe pregnancy complications including severe edema

measurements of gestational sac for dating gestations.

or even progression to seizures related to preE due toBreeding, identification, and dating of pregnancies using

MBG

real-time ultrasound. Squirrel monkey females aretreatment,

clinicalinterventions

includingC-section to end pregnancy may be needed. Veterinarianseasonal breeders with the breeding season for theoversight will be provided throughout the course of thiscolonies in central Texas running from December

trial to minimize threats to maternal well-being.

through April. Beginning in December each year,

MBG treatment in pregnant squirrel monkey: We havefemales enrolled in the trial will undergo ultrasoundmeasured the BP by telemetry device at different

evaluation at weekly intervals until a pregnancy isgestational ages of squirrel monkey. The data haveidentified (typically as a gestational sac, as seen in theshown in Fig. 9.

figure). Then, pregnant females will be examined weekly
during the administration of agents and each two weeks
thereafter until term at about 20 weeks gestation. Theseexams are performed while females are briefly manually
restrained. A typical exam takes 2-3 min., and images are
documented. In the past three years, we have obtained
these measures and developed growth curves for thesquirrel monkey with induced or identified ovulation
dates. These are shown in the growth curves from 45
pregnancies (Fig. 8).
Potential pregnancy interventions in the trial. The
University of Texas MD Anderson Cancer Center animalcare and use program has experience addressing the

Squirrel Monkey Spontaneous Model of Preeclampsia:

occasional need for surgical delivery, post-operative care,

As shown in Fig. 10, the spontaneous model of squirrel

infant re-introduction, nursery rearing if needed, andmonkey and its physical and biochemical features havemanagement of spontaneous labor and delivery in abeen listed.

40

Proceedings of International Symposium on Animal Production and Conservation for Sustainable Development 2018
SESSION 1 - Animal Production and Biotechnology -: 04

Gynecol 192:734-41, 2005.
5. Carter AM. Animal models of human placentation –A Review. Placenta 28:s41-s47, 2007.
6. Chesley LC. Plasma and red cell volumes during
pregnancy. Am J Obstet Gynecol.112:440-450, 1972.
7. Fedorova OV, Kolodkin NI, Agalakova NI, Lakatta
EG, Bagrov AY. Marinobufagenin, an endogenousalpha-1 sodium pump ligand, in hypertensive Dahl
salt-sensitive rats. Hypertension 37:462-466, 2001.
8. Fedorova OV, Simbirtsev AS, Kolodkin NI, et al.
Monoclonal antibody to an endogenous bufadienolide,marinobufagenin,
reverses
preeclampsia-induced
Na/K-ATPase inhibition and lowers blood pressure in
NaCl-sensitive hypertension. J Hypertens 26:2414-2425,
2008.
9. Gallery EDM, Hunyor SN, Györy AZ. Plasma
volume contraction: a significant factor in bothpregnancy-associated hypertension (pre-eclampsia) andchronic hypertension in pregnancy. Q J Med 48:593-602,
1979.
10. Gonick, HC, Ding Y, Vaziri ND, Bagrov AY,
Fedorova
OV.
Simultaneous
measurementofmarinobufagenin, ouabain, and hypertension-associatedprotein in various disease states. Clin Exper Hypertens
20:617-627, 1998.
11. Hays PM, Cruikshank DP, Dunn LJ. Plasma volume
determination in normal and preeclamptic pregnancies.
Obstet Gynecol. 151:958-966, 1985.
12. Heilmann L, Siekmann U, Schmid-Schönbein H,
Ludwig H. Hemoconcentration and pre-eclampsia. Arch
Gynecol 231:7-21, 1981.
13. Hennessy A, Gillin AG, Painter DM, Kirwan PJ,
Thompson JF, Horvath JS. Evidence for preeclampsia ina baboon pregnancy with twins. Hypertension in
Pregnancy 16:223-228, 1997.
14. Horvat D, Severson J, Uddin MN, Mitchell B, et al.
Resibufogenin prevents the manifestations ofpreeclampsia in an animal model of the syndrome.
Hypertens Pregnancy 27:513-711, 2009.
15. Ianosi-Irimie M, Vu HV, Whitbred JM, Pridjian CA,
Nadig JD, Williams MY, Wrenn DC, Pridjian G, Puschett
JB. A rat model of preeclampsia. Clin Exp Hypertens
8:605-617, 2005.
16. Lopatin DA, Ailamazian EK, Dmitrieva RI, et al.
Circulating bufodienolide and cardenolide sodium pumpinhibitors in preeclampsia. J Hypertens 17:1179-1187,
1999.
17. Molvarec A, Szarka A, Walentin S, Szucs E, Nagy
B, Rigo jr J. Circulating angiogenic factors determinedby electrochemiluminescence immunoassay in relation to
the clinical features and laboratory parameters in womenwith pre-eclampsia. Hyperten Res 33:892-898, 2010.
18. Morris JB, Olzinski AR, Bernard RE, Aravindhan K,
Mirabile RC, Boyce R, Willette RN, Jucker BM. P38
MAPK
inhibition
reduces
aorticultrasmall
superparamagnetic iron oxide uptake in a mouse modelof atherosclerosis: MRI assessment. Arterioscler Thromb
Vasc Biol 28:265-271, 2008.
19. Palmer AE, London WT, Sly DL, Rice JM.
Spontaneous preeclampsia toxemia of pregnancy in thepata monkey (Erythorcebus patas). Lab Anim Sci
29:102-106, 1979.
20. Podjarny E, Losonczy G, Baylis C. Animal models

Ethical aspects and research material sharing for the
proposed research. This research proposal uses pregnant
squirrel monkey females and their fetuses with aminimum number of animals per group to obtain valid
results. The acquisition, care, and use of animals will beinaccordance

with

NIH

Guidelines

inan

AAALAC-accredited institution following approval fromthe University of Texas MD Anderson Cancer Center
IACUC. All applicable federal, state, and local laws andregulations will be followed. The most appropriate andhumane form of euthanasia will be used, although thisstudy is not planned as a terminal study. Whereverapplicable, the research protocol will be reviewed andapproved by the institutions’ biohazard committees,
although no Select Agents or recombinant genetic
material is planned for use in these trials. Where possible,
agents available in pharmaceutical grades for humanclinical use will be utilized. Data, images, term infantmonkeys,andfetal/placental tissues

formonkeypregnancies, if produced, will be made available to the
local research community.

References
1. Abi-Ghanem D, Berghman LR, Romo D, Lai X, Li
J, Uddin MN, Kamano Y, Nogawa T, Xu JP, Pettit GR,
Horvat D, Puschett JB. A Chemifluorescent
Immunoassay for the Determination of Marinobufagenin
in Body Fluids. J Immunoassay and Immunochemistry32: 1, 31 – 46, 2011.
2. Agunanne A, Uddin MN, Horvat D, Puschett JB(2010) Contribution of angiogenic factors in a rat modelof pre-eclampsia. Am J Nephrol 32 :332-339, 2010b.
3. Bagrov AY, Fedorova OV. Effects of two putative
endogenous digitalis-like factors, marinobufagenin andouabain, on the Na+, K+-pump in human mesentericarteries. J Hypertens 16:1953-1958, 1998.
4. Buhimschi CS, Norwitz ER Funai E, Richman S,
Guller S, Lockwood CJ, Buhimschi IA. Urinary
angiogenic factors cluster hypertensive disorders andidentify women with severe preeclampsia. Am J Obstet

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of preeclampsia. Semin Nephrol 24:596-606, 2004.
21. Pridjian G, Puschett JB. Preeclampsia, Part I:
Clinical and pathophysiological considerations. Obstet
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22. Puschett JB. The role of excessive volume
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23. Roberts JM, Taylor RN, Musci TJ, et al.
Preeclampsia: An endothelial cell disorder. Am J Obstet
Gynecol.161:1200-1204, 1989.
24. Schoner, W. Endogenous cardiac glycosides, a newclass of steroid hormones. Eur J Biochem 269:2240-2448,
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25. Uddin MN, Horvat D, Glaser SS, Danchuk S,
Mitchell BM, Sullivan DE, Morris CA, Puschett JB.
Marinobufagenin inhibits proliferation and migration ofcytotrophoblast and CHO
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26. Uddin MN, Horvat D, Glaser SS, Mitchell BM,
Puschett JB. Examination of the cellular mechanisms bywhich marinobufagenin inhibits cytotrophoblast function.
J Biol Chem 283:17946-17953, 2008b.
27. Uddin MN, McLean LB, Hunter FA, Horvat D,
Severson J, Tharakan B, Childs EW, Puschett JB.
Vascular leak in a rat model of preeclampsia. Am J
Nephrol. 30:26-33, 2009a.
28. Uddin MN, Horvat D, Childs EW, Puschett JB.
Marinobufagenin causes endothelial cell monolayer
hyperpermeability by altering apoptotic signaling. Am J
Physiol Regul Integr Comp Physiol 296:R1726–R1734,2009b.
29. Uddin MN, Agunanne E, Horvat D, Puschett JB.
Alterations in the renin-angiotensin system in a rat modelof human preeclampsia. Am J Nephrol 31:171-177,
2010a.
30. Uddin MN, Agunanne E, Horvat D, Puschett JB.
Resibufogenin Administration Prevents Oxidative Stressin a Rat Model of Human Preeclampsia. Hypertens
Pregnancy. Dec 21. [Epub ahead of print], 2010b.
31. Uddin MN, Horvat D, DeMorrow S, Agunanne EE
and Puschett JB. Marinobufagenin is an upstream
modulator of Gadd45a stress signaling in preeclampsia.
Biochim Biophys Acta 1812: 49-58. 2011.
32. Vu H, Ianosi Irimie M, Pridjian C, Whitbred J, et al.
Involvement of marinobufagenin in a rat model ofhuman preeclampsia. Am J Nephrol 25:520-528, 2005.
33. Vu H, Ianosi-Irimie M, Danchuk S, et al.
Resibufogenin corrects hypertension in a rat model ofhuman preeclampsia. Exp Biol Med. 231: 215-220,
2006.
34. Ward KW, Proksch JW, Azzarano LM, Salyers KL,
McSurdy-Freed JE, Molnar TM, Levy MA, Smith BR.
SB-239063, a potent and selective inhibitor of p38 Map
Kinase:PreclinicalPharmacokinetics
andSpecies-SpecificReversible
Isomerization.
Pharmaceutical Res18:1336-1341, 2001.
35. Watson LE, Kuo SR, Katki K, Dang T, Park SK,
Dostal DE, Tang WJ, Leppla SH, Frankel AE. Anthrax
toxins induce shock in rats by depressed cardiac
ventricular function PLoS One. 2007 May 23;2(5):e466.
36. World Health Organization (2000) Maternalmortality in 2000; Estimates Developed by WHO,
UNICEF and UNFPA. Geneva, World HealthOrganization, 2004.
37. Wulff C, Weigand M, Kreienberg R, Fraser HM.
Angiogenesis during primate placentation in health anddisease. Reproduction 126:569-577, 2003.

42

Proceedings of International Symposium on Animal Production and Conservation for Sustainable Development 2018
SESSION 2 - Avian Production and Reproduction -: 01

Impact of avian oviduct environment on preservation of mammalian spermatozoa
Ahmed M. Hanafy
Department of Animal Production, Faculty of Agriculture, Suez Canal University, 41522 Ismailia, EgyptCollege of Grassland Science, Gansu Agricultural University, Lanzhou, Gansu province, China

SUMMARY:
The unique features of avian reproduction include the secretion of the uterine fluid (UF) and the presence ofspecialized sperm storage tubules in the female oviduct. Numerous studies have demonstrated that the oviduct provides
suitable environment for the resident sperm without the loss of their viability and fertility. It is reasonable to elucidate
whether deposition of mammalian spermatozoa in the hen oviduct affects on extension the functional lifespan of sperm.
Therefore, fresh rabbit semen were artificially inseminated into the oviduct of laying quail to examine the effect of its
environment on migration and preservation of the sperm over an extended period. Following 0, 10, 30, 60, 90, and 120
min of artificial insemination, the oviduct of quail was dissected from the body cavity and cut into four parts. The
individual segments of the oviduct were excised longitudinally, followed by washing with saline. The mucosal layer of
each part was scraped using a scalpel. The viability of sperm in the washing buffer and mucosal layer was examined
under a phase contrast microscope. Generally, the secretion of avian oviduct may prolong the survival rate and influencefunction of the mammalian sperm. This study has the potential for further development of reliable commercial
extenders that contributes to livestock production and welfare at some time in the future.

Introduction

maintain their survival and fertility potentials over

Artificial insemination (AI) is a powerful tool mostly

prolonged periods. Particularly, their secretions ofutilized for livestock reproductive improvement. It is theuterine fluids (UFs) are composed of energy source,

most common technique in which collected semen canmainly glucose, fatty acids and complex mixture of

be diluted and mixed with an extender to generateorganic and inorganic salts (Ahammad et al., 2013;

hundreds of doses from a single ejaculate. Once the

Huang et al., 2016). These UFs have been found tosemen is diluted, it can be preserved for short periods (3-modulate

5 days) at 5 °C or for long periods of time (years) in

fertilizing capacity of the sperm throughout maintaining

gradually cooled to -195.6 °C using liquid nitrogen

pH and providing a nutritive protective aqueous(Chupin and Thibier 1995). Recently, several extenders

environment in the oviduct (Edward and Leach 1980;

have been developed for liquid storage of semen, but

Holm et al., 2000; Ahammad et al., 2013).In addition,none of them can store spermatozoa for more than 3 days

attachment intimate of sperm within an anatomical

without a decline in fertility (Vyt et al., 2004; El Azazzi

structure, sperm storage tubules (SSTs), in the female

and Hanafy 2015). Even though conventional extenders

reproductive tract is the most important factor forare also used to prevent cell membrane against damages

attaining prolonged sperm storage. Inside SSTs, blind-during cryopreservation, addition of glycerol causes

ended reservoir, the lifespan of fertile sperm issignificant deteriorates in motility and viability of theconsiderably longer, up to 3-5 weeks in domestic fowl

sperm (Aires et al., 2003).

and 10-15 weeks in turkey, depending on the respective

Avian, reptile, amphibian and some fish species arenumbers of SSTs (Bakst et al., 2011). Most of the SSTs

renowned to have a remarkable capability to store

are predominant within the utero-vaginal junction (UVJ)

spermatozoa in female reproductive tract and sustain

mucosal folds that constitute a primary storage sitetheir fertility for long periods, up to months and years,(Bakst 1998). The secondary sperm storage location in

dependent

havethe oviduct is situated within the distal infundibulum

demonstrated that the oviduct of birds provide anwhich is characterized by sub-epithelial tubular glandenvironment appropriate for the resident sperm to(Fujii and Tamura 1963). Although the exact approach byon

species.

Numerous

studies43

motility,

extendlifespanandmaintainProceedings of International Symposium on Animal Production and Conservation for Sustainable Development 2018
SESSION 2 - Avian Production and Reproduction -: 01

which spermatozoa are preserved in the avian oviductintra-vaginally introduced to the cloaca of female quailhas not been completely clarified, abundant studies haveusing a plastic dropper. At the time of AI, the female with

been carried out to evaluate the environmental features

the presence of an egg in the uterus, ascertained byof the oviduct and concluded their utility on resident

palpation, was unused. Following 0, 10, 30, 60, 90, andsperms. Therefore, the aim of this study is to determine

120 min of a single insemination, the oviduct of thethe role of quail oviduct environment on sperm survival

female quail (n=3 per each time) was removed from theof mammalian species. Three questions were postulated

body cavity after slaughter and cut into four segments,

as follows: (1) whether rabbit sperm able to migrate in

infundibulum, magnum, isthmus and uterus. The

the oviduct of quail; (2) whether motility and viability of

individual parts of oviduct were excised longitudinally,

deposited sperm affects by environment of quail oviduct;

followed by washing for two times with 10ml of theand (3) whether the mammalian spermatozoa can bindisotonic solution (0.9% NaCl) in Petri dishes. The

within SST of the avian oviduct.

washing solution was collected and centrifuged at 800× g

Material and Method

for 5 min to precipitate and the final pellet was thenAnimals

resuspended in 1 ml of saline solution for spermatozoa

Mature female Japanese quails (Coturnix japonica), aged

observation. The mucosal layer of each segment was

20 weeks with proven fertility, were used in this study.

scraped using a scalpel. Viability of the sperm in

At least one month before insemination, eighteen female

washing buffer and in mucosal layer was examined

quails, close in body weight (251.91±2.11g) and egg

under a phase contrast microscope at 600× magnification.

production rate (76.00±1.14%), were separated from

Results and Discussions

male

under

Sperm motility and viability are the main factor affecting

photoperiod regimen of 16L: 8D. Experimental birds

sperm function for migration in the oviduct. As shown inwere fed laying ration (20% crude protein (CP) and 2900

Table (1), all obtained values of ejaculate volume, sperm

kcal ME/kg) and supplied with water ad libitum.

cell concentration, progressive motility, and sperm

One-year-old New Zealand White rabbit (Oryctolagus

viability of pooled ejaculates immediately before AI were

cages

and

maintained

individuallycuniculus) bucks (3.98 ± 0.1 kg body weight) were used

Table (1). Means ± SE of initial characteristics offor semen collection. Two mature bucks were housed insemen samples collected from NZW rabbit bucks.

flat-deck cages with semi-opened system. Animals were

Parameters

fed a commercial standard diet that contains 18 % CP and

Ejaculate volume (ml)Concentration (× 108/ml)
Progressive motility (%)
Sperm viability (%)

2700 kcal ME/kg. Drinking water was automaticallyoffered freely.
Semen collection and evaluation
Fresh semen of rabbit bucks with normal sperm quality

in normal ranges of rabbit semen characteristics.

Immediately after collection, good quality ejaculates were

In this study, the ability of mammalian spermatozoa tomixed together and treated as one pooled semen sample.

navigate and survive within avian oviduct has been

Sperm motility was assessed by visual estimation using aobserved. As shown in Table (2) and Fig (1), motile

phase contrast microscope (Olympus, Japan). The sperm

sperms were observed in the uterus and isthmus 10 min

concentration was determined using a Bürker counting

after AI. No sperm could be found in the uterine cavity

chamber and light microscopy at 400× magnification. For

60 min after AI, demonstrating the importance of thedetermination of progressive motility, a drop of 10 l ofoviduct cavity for assisting the transport of rabbit sperm

semen was delivered onto a clean glass slide, covered with

into the infundibulum of quail. From this result, it could

a cover slip, and estimated at 600× magnifications.
spermatozoa

wereexamined

0.58 ± 0.10
3.94 ± 0.21
78.18 ± 0.72
93.01 ± 0.59

SE= Standard error(Table 1) was collected by an artificial vagina.

Live/dead

Mean ± SE

be proposed that the uterine fluids (UFs) did not harmonthe viable and motile sperm of rabbit (Ahammad et al.,Eosin-Nigrosine-based stains smears using a phase contrast2013). While immotile sperms were detected in uterus

microscope. Prior to insemination, spermatozoa were kept

and isthmus after 30 min, it was still survived inat room temperature in darkness.

magnum and infundibulum. After one hour of AI, motile

AI of female quail

sperm has only been observed in the infundibulum, but

For AI, 0.1 ml of undiluted fresh semen of rabbit was44Proceedings of International Symposium on Animal Production and Conservation for Sustainable Development 2018
SESSION 2 - Avian Production and Reproduction -: 01

immotile sperm was detected at the lower parts of theselection in the storage sites. Moreover, intact acrosome

oviduct. Although immotile sperms were found in themembrane was observed 1 h after AI in infundibulum

magnum and infundibulum at 90 and 120 min ofpart of the oviduct (Fig. 1). The percentage of

insemination, no motile or immotile sperms were

spermatozoa that able to keep the intact acrosome canobserved in the other segments of the oviduct. These

give an indication on the fertilizing capacity of sperm

results showed that within a few minutes of semen(Richardson et al., 1991). Prepare rabbit sperm todeposition, rabbit sperm begin to migrate into the upper

undergo the acrosome reaction relatively slowly is aparts of the quail oviduct. Although spermatozoa

significant factor that necessary for fertilizing success.

oriented themselves in the oviduct, it is not yet known

Table (2). Description of presence and viability ofrabbit spermatozoa in washing buffer collectedfrom segments of quail oviduct after IA.
Infundibulum
Time Uterus Isthmus Magnum
0
N
N
N
N
10
M
M
N
N
30
IM
IM
M
M
60
N
IM
IM
M
90
N
N
IM
IM
120
N
N
IM
IM

whether thermotactic, mechanotactic and/or chemotactic
mechanisms guide sperm to transport throughout theoviduct. It has been reported that rabbit sperm tend to
swim towards warmer temperatures in the reproductive
tract of inseminated does (Bahat et al., 2003).
Table (3). Description of presence and viability ofrabbit spermatozoa in mucosal layer collected fromuterus and infundibulum of quail oviduct after AI.
UterusInfundibulumTime(min)
0
N
N
10
N
N
30
M
N
60
M
M
90
M
M
120
M
M

M= motile sperm; IM= immotile sperm and N= no sperm wasfound.

To our knowledge, no information is published before on
the viability and motility of mammalian spermatozoa
entering the female reproductive tract of birds. Therefore,
it might be interesting to evaluate whether rabbit sperm
can bind within SST pockets of female quails and

M= motile sperm and N= no sperm was found.

consequently prolonged flagellar activity and viability of

It is worthy noted that SST not only act as a sperm

spermatozoa. As shown in Table (3) and Fig (2), rabbit

storage site but may also play a role in selection of sperm

spermatozoa have the ability to interact and intimatethat will be preserved. Based on the present findings,

associate within SST in the uterus and infundibulum. In

most of motile sperm which identified in the washing

contrast, no sperm storage site could be detected in thesolution appear to be abnormal motile spermatozoa. This

isthmusfinding doesn’t eliminate the possibility of sperm

spermatozoa was higher in the infundibulum than theormagnum.

The

distribution

of

bind

uterus segment 2 h after AI. Nevertheless, all heads ofthe sperm that were detected in the segments were
extremely embedded in the SST, which allowed only the
motile tails of these spermatozoa to be clearly visible.
While mammalian sperm interacts with the surface ofepithelial cells at the utero-tubal junction (Rijsselaere etal., 2004); Ito et al. (2011) speculated that resident sperm
in the oviduct of quail “seems to be free from theepithelial cells of the SST” and most of them weresluggish. However, it is unclear whether the flagellar
activity of buck sperm was related to the characters ofsperm or to the technique of the study since the previous

Fig 1. Scanning light microscope showing motile (A) and immotile(B) sperm of rabbit in washing buffer of oviduct segments after
AI. After AI, uterus (C) and infundibulum (D) of female quails
were washed at 10 and 60 min, respectively; and motile sperm
was observed by a phase contrast light microscope at 600×
magnification. While arrowhead, thick and thin arrows in A were
showed the head, midpiece and flagellum of the sperm,
respectively, small arrows in D were identified the acrosome area.

authors (Ito et al., 2011) generated an in vitro study inquail to determine the flagellar movement in the SST.
Because there are no eggs in the oviduct of used female
quails, it is acceptable to presume that introduced sperm
can reach the site of fertilization without an obstacle

45

Proceedings of International Symposium on Animal Production and Conservation for Sustainable Development 2018
SESSION 2 - Avian Production and Reproduction -: 01

from the downward egg. There is positive evidence that

communities.

sperm quantity and motility are crucial for SST

Acknowledgement

permeation (Froman et al., 2013). The number of sperm

The author thanks Mrs. Islam Abd El-kader and Azaam

inseminated in this study may be more than that of native

Oraby for their technical assistances and Dr. Ibrahim M.

condition (Hanafy and Khalil 2015). Hence, the quantity,

Hegab (Suez Canal University) for critical reading andas well as quality, of spermatozoa plays a critical role in

English editing of the manuscript.

sperm entering and persistence in the SST. However,

Reference

how sperm involved within SSTs and under which

1) Ahammad MU, Miyazato T, Nishino C, Tatemoto H,
Okura N, Okamoto, Kawamoto Y S, and Nakada T(2013) Effects of fluid secreted from the uterus on
duration of fertile egg production in hens, andsurvivability and penetrability of fowl sperm in vitro.
Journal of Poultry Science, 50:74-82.

mechanisms still an unanswered question.

2)Aires VA, Hinsch KD, Müller-Schlösser F, Bogner K,
Müller-Schlösser S and Hinsch E (2003) In vitro and invivo comparison of egg yolk-based and soybean
lecithin-based extenders for cryopreservation of bovine
semen. Theriogenology, 60: 269-79.
3) Bahat A, Tur-Kaspa I, Gakamsky A, Giojalas LC,
Breitbart H and Eisenbach M (2003) Thermotaxis of
mammalian sperm cells: a potential navigation
mechanism in the female genital tract. Nature of
Medicine, 9:149–150.
4) Bakst MR (1998) Structure of the avian oviduct withemphasis on sperm storage in poultry. The Journal of
Expermental Zoology, 282:618–626

Fig 2. Scanning light microscope showing rabbit spermatozoa
attached to SST in infundibulum (A and B) and uterus (C and D)of quail after 2 h of AI. Arrow indicates the SST and arrowheadshows the flagella of resident sperm in the SST. A representative
photograph was detected by phase contrast light microscope at
600× magnification.

5) Bakst MR (2011) Role of the oviduct in maintaining
sustained fertility in Hen. Journal of Animal Science,
89:1323–1329

On the other hand, immune system of female quail is

6) Chupin D and Thibier M (1995) Survey of the present
status of the use of artificial insemination in developed
countries. World Animal Review, 82:58-68.

particularly active in the oviduct for protection against
microbes and may also attack sperm as foreign bodies

7) Das SC, Nagasaka N and Yoshimura Y (2005b)
Changes in the localization of antigen presenting cells
and T cells in the utero-vaginal junction after repeatedartificial insemination in laying hens. Journal of
Reproduction and Development, 51:683–687.

(Das et al., 2005b). Normally, oviductal cellular immune
responses are suppressed in the SST of chicken toconserve storage sperm (Das et al., 2006b). Whileoviduct of quail might be considering sperm of rabbit as8) Das SC, Isobe N, Nishibori M and Yoshimura Y(2006b) Expression of transforming growth factor-ȕ
isoforms and their receptors in utero-vaginal junction ofhen oviduct in presence or absence of resident sperm withreference to sperm storage. Reproduction, 132: 781–790

an unfamiliar body, the invasion may take time to build
an effective level of immune response. Indeed, the motile
sperm of rabbit may not harm by leukocytes of does that
increase after coitus in the reproductive tract of thefemale (Taylor 1982). Altogether, even though this9) Edward SJr and Leach RMJr (1980) Studies on theorganic components of shell gland fluid and the hen’s egg
shell. Poultry Science, 59:438-443.

speculation at present is an imaginative idea; it is at least
based on biological findings.

10) El-Azzazi FE and Hanafy AM (2015) Impact ofsoy-lecithin based extender on quality and fertility ofpreserved rabbit semen. Egyptian Journal of Rabbit
Science, 25:197 – 209.

Conclusion
From this preliminary study three conclusions could be
withdrawn: (i) 2 h after AI, spermatozoa of rabbit weremainly found in the SST of the uterus and infundibulum,11) Froman DP and Rhoads DD (2013) Breeding andgenetics symposium: A systems biology definition forchicken semen quality. Journal of Animal Science, 91:
523-529.

(ii) the environment of oviduct influenced deposited
sperm transport in the female genital tract of quail, and(iii) this result appeared to be open a wide discussion
more than answering few questions for scientific46Proceedings of International Symposium on Animal Production and Conservation for Sustainable Development 2018
SESSION 2 - Avian Production and Reproduction -: 01

12) Hanafy AM and Khalil HA (2015) Influence of chronic
Dexamethasone administration on Reproductive
parameters and semen traits in male of Japanese quail.
Asian Journal of Poultry Science, 9: 223-232.

17) Richardson BS, Nikolajczyk LH, Abdullah JC and
O’Rand MG (1991) Localization of rabbit sperm acrosin
during the acrosome reaction induced by immobilized
zona matrix. Biology of Reproduction, 45:20-26.

13) Fujii S and Tamura T (1963) Location of sperms in theoviduct of the domestic fowl with special reference tostorage of sperms in the vaginal gland. Journal of Facultyof Fisheries and Animal Husbandry, Hiroshima
University, 5:145–163.

18) Rijsselaere T, Van Soom A, Maes D, Verberckmoes,de and Kruif A (2004) Effect of blood admixture on invitro survival of chilled and frozen-thawed canine
spermatozoa. Theriogenology, 61:589-1602.
19) Taylor NJ (1982) Investigation of sperm-induced
cervical leukocytosis by a double mating study in rabbits.
Journal of Reproduction and Fertility, 66:157–160.

14) Holm L, Ekwall H, Wishart GJ and Ridderstrale Y(2000) Localization of calcium and zinc in the sperm
storage tubules of chicken, quail and turkey using X-ray
microanalysis. Journal of Reproduction and Fertility, 118:
331–336.

20) Vyt P, Maes D, Dejonckheere E, Castryck F, Van
Soom A (2004) Comparative study on five different
commercial extenders for boar semen. Reproduction in
Domestic Animals, 39:8-12.

15) Huang A, Isobe N, Obitsu T and Yoshimura Y (2016)
Expression of lipases and lipid receptors in sperm storage
tubules and possible role of fatty acids in sperm survivalin the hen oviduct. Theriogenology, 85:1334-1342
16) Ito T, Yoshizaki N, Tokumoto T, Ono H, Yoshimura
T, Tsukada A, Kansaku N and Sasanami T (2011)
Progesterone is a sperm-releasing factor from thesperm-storage tubules in birds. Endocrinology, 152:3952-3962.

47

Proceedings of International Symposium on Animal Production and Conservation for Sustainable Development 2018
SESSION 2 - Avian Production and Reproduction -: 02

Possible roles of the yolk sac as a source of thyroid hormones during embryonicdevelopment of the chicken
Hanny Cho Too
Livestock Breeding and Veterinary Department, Myanmar

SUMMARY
In general, thyroid hormones (THs) are essential for the correct development of nearly every
structure in the body from very early stages of development, yet the embryonic thyroid gland is not
functional at these stages. To clarify the role of the egg yolk as a source of THs, I have analyzed the TH
content in the yolk and the expression levels of the D1, D2, D3, TTR, and ALB gene in the same animalthroughout the 21-day incubation period to gain a broad-range view of the roles of the yolk sac membranein processing and supplying THs to the developing embryo. In addition, the expression of MCT8, MCT10,
and OATP1C1 gene which are assumed to be responsible for transport of yolk THs in and out of the yolk
sac membrane, was determined. To summarize, the expression of D3 and TTR in the earlier period, D2 and
OATP1C1 in the later period, and D1, ALB, MCT8, and MCT10 were expressed in both periods ofincubation.
and they regulate intracellular T3 levels in a tissue-

Introduction
In all vertebrate species and invertebrate species so

specific way. But the most important enzymes for TH

far, thyroid hormone (TH) is significant for regularmetabolism are the iodothyronine deiodinases. The

functioning of almost all body’s organs. The thyroid

early embryo does not have a thyroid function andcontains

and

therefore it depends on the THs supplied by maternal

triiodothyronine (T3). T4 is the main hormone released

hormone effect on the yolk of the chicken (Stijin et al.,from the thyroid glands and it is transformed into2012). Some studies indicated that thyroid hormones

biologically active T3. Intracellular activation or

are present in the egg yolk of chicken and quail but

inactivation of T4 and T3 is determined by three types

most of studies are focused on the late period ofof idothyronine deiodinases such as D1, D2, and D3

incubation.

(Gereben et al., 2008). THs are transported throughout

lipoproteins and enters embryo with non-specificthe body via the circulation. The major TH distributor

uptake of yolk or by specific carrier (Mcnabb et al.,in birds and other non-mammalian vertebrates are1997). In mammals, thyroid hormone reaches to thetransthyretin (TTR) and serum albumin (ALB). TTR isembryo by the potential effects of maternal thyroid

one of the proteins in extracellular fluids, which binds

hormones without crossing the placenta but in rats,

the thyroid hormones T4 and T3. Visser et al., 2008

some amounts of thyroid hormones do across therevealed that transport of T4 and T3 in and out of cells

placenta (Fisher et al., 1977; Roti et al., 1983). Someis controlled by several classes of transmembrane

studies indicated that thyroid hormones are present

transporter, including members of the organic anion

primarily in the eggs yolk of chicken and quail, 4-6 ngtransporters family (OATP), L-type amino acidT4/g and 1.5-2.5 ng T3/g of yolk ((Mcnabb et al.,transporters (LATs) and monocarboxylate transporters

1997). Therefore, the objectives of my study are to(MCTs). Thyroid hormone transporters are necessary

observe the changes of iodothyronine deiodinase

for the uptake of thyroid hormone into target tissues

mRNA expression, carrier proteins and TH transporters

two

hormones

thyroxin(T4)

48

Thyroidhormone

bound

to

yolk

Proceedings of International Symposium on Animal Production and Conservation for Sustainable Development 2018
SESSION 2 - Avian Production and Reproduction -: 02

for transporting THs to the embryo before and after theal., 1997. T3 and T4 were measured using enzyme

onset of thyroid function. In addition, yolk TH content

immunoassay kits (Immunospec Corporation, Canoga

was examined by enzyme immuno assay.

Park, CA, USA). According to the manufacturer’s

Material and Method

document, the cross-reactivity with related compounds

Animals and Samplingother than T3 and T4 is less than 1.5%. The linearity of

Fertilized eggs from layer strain (Hy-line) chicken

the yolk extract and the serum sample to the standard

were purchased from Japan Layer K.K., Gifu, Japan.

curve was confirmed by measuring serially samples

The eggs were incubated at 37.8 Ԩ and 70% relativewith the assay buffer provided in the immunoassay kit.

humidity. The day of onset of incubation was labeled

The inter-assay variations were 6.2-12.4% and 3.8-

embryonic day 0 (E0). All the embryos (chicks) were

12.9%, respectively.

piping with their beaks outside the eggshell at the time

Results and Discussionsof E21 sampling. The yolk sac membrane was sampled

The present results indicated that the yolk weightdaily from E4 embryos to 3-day-old chicks (C3) (n=7-decreased significantly and almost linearly during the9). The membrane was rinsed thoroughly with sterile

incubation period (Fig. 1). As shown in Fig. 1, the total

phosphate-buffered saline (PBS) with several changes

yolk TH content decreased in comparable tendency,

to remove remaining yolk, put on a piece of filter paper

and thus the yolk is likely to supply its components,

to absorb excess PBS, weighed on an electronic

including THs, to the developing embryo. The present

balance, andthen immediately frozen in liquid

study showed that the expression of TTR, whose

nitrogen and stored at -85Ԩ until RNA extraction. The

protein has high affinity for THs, was very low in theyolk contents were collected every other day (n=5), putyolk sac membrane from E13 onwards (Fig. 2B). A

into plastic tubes, weighed on an electronic balance,

possibility is that ALB, whose gene expression

and stored at -30Ԩ for measuring the yolk TH content.

increases between E1 and E18, contributes to TH

All animal manipulations were approved by the Animal

distribution to the embryo in turn (Fig. 2B). These

Research and Welfare Committee at Gifu University.

results seem to indicate that the yolk contains a

Quantification of mRNA expression

sufficient amount of THs that can be supplied to the

Total RNA was extracted from frozen yolk sacembryo even after the onset of thyroid function. It ismembrane using TRIzol reagent (Invitrogen, Carlsbad,

assumed that TH inactivating D3, whose gene was

CA, USA). RNase-free DNase I (gDNA Remover,expressed in almost the same period as TTR and ALB

TOYOBO Co. Ltd., Osaka, Japan) was used toE14-E12, could regulate the amount of TTR and ALB-

eliminate

theborne T3, thereby preventing unfavorable premature

manufacturer’s instructions. Total RNA (1ʅg) was thenTH signaling in the earlier embryo. The expression ofreverse transcribed into first-strand cDNA using

D2, the TH-activating deiodinase gene, was highReverTra Ace (TOYOBO) and a random primer

between E14 and E21. This period corresponded to the(TOYOBO). The mRNA levels were determined byincrease of circulating T4, beginning at around E14 andreal-time PCR, performed in an Mx3000P Real-Timereaching its peak on E20. The expression of OATP1C1,

PCR system (Agilent Technologies, Santa Clara, CA,

the gene of transmembrane transporter, was also highUSA) with the two-step standard cycling program forat around this period (Fig. 2C). It may be responsible

the Brilliant III Ultra-Fast SYBR® Green QPCR Masterfor transporting plasma T4 into the yolk sac epithelialMix (Agilent).

cells. The D1 gene expression was expressed almost

Measurement of yolk THs by enzyme immunoassay

throughout the 21-day incubation period (Fig. 2A).

genomic

DNA

accordingto

THs were extracted from the frozen yolk using theThus, D1 in the yolk sac membrane could contribute tomethanol/chloroform extraction procedure of Wilson et

the recycling of iodine (by 5’ –deiodination of rT3 to49

Proceedings of International Symposium on Animal Production and Conservation for Sustainable Development 2018
SESSION 2 - Avian Production and Reproduction -: 02

D1 mRNA ( V.S. 18S)

0.06

than to the activation of T4 to T3.

3.5
2.5

25
20

2

15

1.5

10

1

5

0.5

D3

0.04

0.15
0.1

0.03
0.02

0.05

0.01

0

0

incubation period
Fig .2 (A)00E3 E5 E7 E9 E11 E13 E15 E17 E19 C1

TTR mRNA (V.S. 18S)

12

incubation period

Fig.1. Yolk thyroid hormone content during embryonicdevelopment of the chicken by enzyme immune Assay.
Each data presents mean ± SEM (n=5).

TTR
ALB

10
8
6
4
2
0

1.4
1.2
1
0.8
0.6
0.4
0.2
0

ALB mRNA (V.S. 18S)

yolk weight(g)3

Yolk T4 and T3(ng/egg)

yolk weight
T3
T4

0.2
D1

0.05

D3 mRNA( V.S. 18S)

T2) that cannot be supplied from outside the egg, rather

Conclusions
incubation period

First, present results have confirmed previous
MCT8 mRNA (V.S. 18S)

available to the embryo (Mcnabb et., al. 1997).
Second, this study has shown that yolk THs could be
metabolized before their distribution to the embryo or
action in the yolk sac membrane. A part of the maternal
THs contained abundantly in the yolk is probably

0.4
0.35
0.3
0.25
0.2
0.15
0.1
0.05
0

MCT8
OATP1C1

0.9
0.8
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0

inactivated by D3, preventing unfavorable premature
T3 signaling. The resulting rT3 could easily be

incubation period

deiodinated further by D1, which can be contributed toFig. 2 (C)OATP1C1 mRNA (V.S. 18S)

Fig. 2 (B)observations that maternal THs in the yolk are

the recycling of iodine. The present study suggests thatFig.2. D1, D3, TTR, ALB, MCT8 & OATP1C1 mRNA
expression levels in the yolk sac membrane during
embryonic development of the chicken by RT-PCR. (18S
was used as an internal control) (IP=internal pipping)
Each data presents mean ± SEM (n=7).

the yolk sac membrane not only transfers yolk THs tothe embryo but also activates THs, thereby meeting thedemands of the developing embryo and the yolk sacmembrane.

Acknowledgements
I would like to express my sincere gratitude to my

References

supervisor Professor IWASAWA Atsushi, Laboratory

Denver, R.J. (1993) Acceleration of anuran amphibianof Comparative Biochemistry, Gifu University, whometamorphosis

has been supportive of this work. My sincere thanks

bycorticotrophin-releasing

hormone-like peptides. Get. Comp. Endocrine. 91:

also go to SHIBATA Mitsuhiro, United Graduated38-51.

School of Agricultural Science, for his kind support on

Fisher, D. A., J. H. Dussault, J. Stack, and I. J. Chopra

this work and technical assistance.

(1977) Ontogenic of hypothalamic-pituitary-thyroid
function and metabolism in man, sheep and rat. Rec.
Progr. Horm. Res. 33:59-116

50

Proceedings of International Symposium on Animal Production and Conservation for Sustainable Development 2018
SESSION 2 - Avian Production and Reproduction -: 02

Gereben, B, Zeold, A., Dentice M., Salvatore, D.,
Bianco, A.C (2008) Activation and inactivation of
thyroid hormone by deiodinases: local action withgeneral consequences. Cell. Mol. Life Sci. 65: 570590
Mcnabb F.M.A and Wilson C.M. (1997) Thyroid
hormone deposition in avian eggs and effects onembryonic development. Amer Zool., 37:553-560
Wilson C.M, McNabb F.M. (1997) Maternal thyroid
hormones in Japanese quail eggs and their influence
on embryonic development. Gen Comp Endocrinol
107: 153-165.
Roti, E., A. Gnudi, and L.E. Braverman (1983) The
placental transport, synthesis and metabolism ofhormones and drugs which affect thyroid fucnction.
Endocrine Rev. 4:131-149
Stijin L.J. Van Herck, Stijin Getsens, Joke Delbaere,
Przemko Tylzanowski, Veerle M. Darras (2012)
Expression

profileandthyroidhormoneresponsiveness of transporters and deiodinases inearlyembryonic

chickenbrain

development.

Molecular and cellular Endocrinology 349: 289-297
Visser. W.E., Friesema, EC., Jansen, J., Visser, T.J(2008) Thyroid hormone transport in and out ofcells. Trends Endocrinol. Metab. 19: 50-56

51

Proceedings of International Symposium on Animal Production and Conservation for Sustainable Development 2018
SESSION 2 - Avian Production and Reproduction -: 03

Comparative studies of the morphology of oviduct of pre-laying, laying and regressing
chickens from indigenous, sonali and RIR breeds (Gallus domesticus L.)
Md. Anisur Rahman*, Mst. Shamima KhanamDQG Mst. Sabiha Hossain

Department of Zoology, University of Rajshahi, Bangladesh. *Email: ar_zool.uk@fastermail.com

SUMMARY
The morphological measurements of length (cm) and weight (gm) of total oviduct from each reproductive stage (prelaying, laying and regressing) of every breed (indigenous, sonali and RIR) were determined. The lengths (mean ± sd) of
total oviduct for indigenous breed were 22.63±7.57cm (pre-laying), 50.76±2.17cm (laying), 44.93±9.37cm (regressing);
for sonali were 9.53±1.05cm (pre-laying), 73.16±6.40cm (laying), 53.76±5.67cm (regressing) and for RIR were
13.53±0.73cm (pre-laying), 91.26±4.23cm (laying), and 83.93±2.48cm (regressing). The weights (mean ± sd) of total
oviduct were 4.56±1.55gm (pre-laying), 32.30±1.94gm (laying), 19.83±7.65gm (regressing) for indigenous;
2.77±0.63gm (pre-laying), 51.53±7.52gm (laying), 27.90±13.64gm (regressing) for sonali and 3.70±1.90gm (prelaying), 93.26±6.39gm (laying), and 59.30±2.44gm (regressing) for RIR breeds, respectively.
The mean values (F) of length of oviduct of each stage from different breeds were significantly different (F3,16=31.49,
pre-laying; F3,16=12.29, laying; and F3,16=11.06, regressing; (P<0.05)). Similarly, the mean values (F) of weight weresignificantly different (F3,16=43.52, pre-laying; F3,16=7.44, laying and F3,16=13.50, regressing; (P<0.05). The
reproductive fitness in chicken is assessed by the examination of oviductal parameters and this study is very much
important in reproductive biology for the commercial egg and meat production through improved breeding plansessential to conserve the endangered birds from their extinction.
the morphological knowledge of reproductive tract isIntroduction
very much essential to the commercial producers and
Rapid growth of human population is a burning issue
breeders for the production of egg and meat productive
in developing countries like Bangladesh. The
chicken. The disrupted activity or pathological changes
insufficient animal protein supply to increasing
can alter or deviate the function of the oviduct of a
populations necessitates the increase of animal
laying hen directly affects egg and eggshell quality
productivity which is on the other hand very much
(Chousalker & Roberts, 2008). Decline in egg and egg
important for the conservation of endangered animals
shellquality costs the egg industry millions of dollars
considering animal and human welfare. Poultry meat
every
year (Abdel-Rahman et al., 2016) and ultimately
contributes 37 percent of total meat production and 22
decrease
economic profitability (Chousalker andto 27 percent of the total animal protein supply in the
Roberts, 2008).
country (FAO, 2003). Annual per capita egg
consumption was 32 eggs (Das et al., 2008), reached its
The avian oviductal tissues undergo dynamic
highest level of 48 eggs in 2012 with a deficit ofmorphological and functional changes during
53.85% (FAO, 2015) compared to the minimum
reproductive cycle. The most important processes
requirement of 104 eggs/head/year and meat
involved in avian oviduct development and function
consumption was 2.92 kg/year to the minimum 7.67
seem to be extensive cell proliferation andkg/year (Begum, 2008). To reduce the deficiency, thedifferentiation of the luminal and glandular epithelium
gap between the continuous demand and short supply
in Japanese quail (Rahman et al., 2007, 2009) and inof animal proteins could be minimized throughchicken (Jeong et al., 2013; Hrabia et al., 2014). The
increasing chicken egg and meat production. The
morphometrical changes of oviduct at different
chicken egg and meat are used as an alternative amongdevelopmental stages involved in the egg formation.
other sources of animal proteins because of their easy
The avian oviduct has been studied extensively inavailability, low cost, high nutritional values andlaying stage of different birds but a comparative
arbitrary choice.
analysis at different reproductive stages of different
breeds is still in wanting. The length and weight of
The oviduct is a highly complex and dynamic organ
oviduct increased from pre-laying to laying andwith its coiled tubular structure in the reproductive
decreasedfrom laying to regressing stage in chickens
system of avian species where ovulated ovum either
and thus this study provides the information for the
being fertilized or not developed into egg. During theproper understanding and identification of normal
long journey through the entire oviduct, constituents ofreproductive tract related to egg and meat production inegg from the infundibulum, magnum, isthmus, uterus
chicken breeds. Therefore, the present work wasand vagina (Rahman, 2013) are sequentially secreted
designed to study the comparative morphology ofand deposited as layers around the ovulated ova
oviduct of indigenous, sonali (crossbred; RIR ƃX(Rahman et al., 2007) is a necessary prerequisite
Fayoumi Ƃ) and exotic RIR (Rhode Island Red)for ƍnormalƍ egg formation (Solomon, 2002). This egg
chicken breeds in their pre-laying, laying andproduction is of special interest to the commercial egg
regressing reproductive stages.
industry or producers (Chousalker and Roberts, 2008).
Tremendous changes in the size of the chicken oviduct
Material and Method
occurred during laying stage (Yu et a., 1972) indicating
All procedures were approved by Animal Ethicalfunctional and healthy oviduct which is essential for
Committee of Rajshahi University (390/320/IBSc). A
both quantity and quality of egg (Saino et al., 2002). So,
total of twenty seven female chickens from three52

Proceedings of International Symposium on Animal Production and Conservation for Sustainable Development 2018
SESSION 2 - Avian Production and Reproduction -: 03

chicken breeds (indigenous, n=9; crossbred sonali,
n=9; and exotic RIR, n=9) were used for this study.
Each breed was with three reproductive stages and they
were pre-laying (n=3), laying (n=3) and regressing(n=3). These chickens were purchased from village
markets (indigenous) and nearby farms (sonali andRIR) close to Rajshahi University. The chicken age
ranged from 6-10 months for indigenous (pre-laying=6,
laying=8, regressing=10); 3-19 months for sonali (prelaying=3, laying=6, regressing=19) and 4-18 months
for RIR (pre-laying=4, laying=10, regressing=18).
They were allowed to acclimatize for two weeks withwater and layer feed ad libitum and slaughtered byusing halal method in the Genetics and Molecular
Biology Laboratory. The length (cm) and weight (gm)measurements were taken by linear scale and electronicbalance after the removal of whole oviducts from theexperimental hens. Descriptive statistics and one-way
analysis of variance (ANOVA) were subjected toevaluate morphological differences (mean±sd of length
and weight) of each reproductive stage from three
breeds and all statements of significance were assessedat P < 0.05.

comparatively less than the laying ones and theydiffered (F3,16=13.50, P<0.05) from each other in three
breeds (19.83±7.65gm for indigenous at 10 months,
27.90±13.64gm for sonali at 19 months and59.30±2.44gm for RIR at 18 months).
Table 1 Length (cm) and weight (gm) of oviducts frompre-laying, laying and regressing reproductive stages ofindigenous, sonali and RIR chicken breeds.
MorphologicalParameters

BreedsIndigenous
SonaliRIR
F values (P<0.05)
Indigenous
Weight of
Sonalioviduct (gm)
RIR
F values (P<0.05)
Length ofoviduct (cm)Pre-laying(Mean±Sd)22.63±7.57
9.53±1.05
13.53±0.73
F=31.494.56±1.55
2.77±0.63
3.70±1.90
F=43.52

Reproductive Stages
LayingRegressing(Mean±Sd)(Mean±Sd)50.76±2.1744.93±9.37
73.16±6.40
53.76±5.67
91.26±4.23
83.93±2.48
F=12.29
F=11.06
32.30±1.94
19.83±7.65
51.53±7.5227.90±13.64
93.26±6.3959.30±2.44
F=7.44
F=13.50

There were differential growth patterns due todeposition of cellular components from the oviduct atvarious stages during a reproductive cycle (Yu et al.,1972) resulting morphological changes in length andweight of reproductive stages from different breeds
were compared through statistical analyses in thepresent study (Table 1). The total oviduct length andweight were lowest in pre-laying stages of all threechicken breeds and rapidly increased as a result of
rapid development and growth of oviduct in laying
stage and decreased in a similar manner during
regression of the oviduct. The indigenous pre-laying
chicken oviduct mean length of the present study at 6
months 22.63±7.57cm was higher than the mean lengthof 8.73 ± 0.27cm for 3 months and 9.93 ± 0.41cm for 5
months (Rahman et al., 1999) whereas it was lower
than the 59.73 ± 0.85 cm at 5-6 months in local uttara
fowl (Khan et al., 2017). The present mean weight
4.56±1.55gm for 6 months showed similarity with thereport (6.34±1.54gm at 4 months Hyline layer) of
Hrabia et al. (2014) though it was lower than theweight (31.73 ± 0.96gm) of local uttara fowl of 5-6
months (Khan et al., 2017). The sonali crossbred(Ambar et al., 1999) is a cross-breed RIFI (RIR male X
Fayoumi female) introduced in Bangladesh for well
adaptation to the environment (FAO, 2015). The
productivity of sonali was more profitable compared to
RIR and Fayoumi (Rahman et al., 1997), improved
only in egg production in reciprocal cross FIRI
(Rahman et al., 2004) whereas Khawaja et al. (2013)
reported that FIRI chickens were better performer in all
traits than RIFI. To our knowledge the oviductal length
measurement was lacking in pre-laying stage of sonali
breed and in the present study we found its mean
length 9.53±1.05cm at 3 months age. The mean weight
2.77±0.63gm of this study was compatible with theoviduct weight (5.05gm) of the pre-laying stage (4months) of the commercial hybrid Dekalb strain (Yu etal., 1972). RIR birds at 5 months had oviduct with 22.7
± 0.727cm length and 18.5 ± 0.485 gm weight(Banerjee et al., 2006) and for same age pre-laying
showed 32.21cm length and 22gm weight (Singh,2001). ISA brown chicken oviduct length increased
depending on age (3months, 9.97±0.27cm; 5 months,
11.33±0.32cm) (Rahman et al., 1999). The present
findings bearing the mean length at 4 months

Result and Discussions
The results for mean lengths (cm) of the oviducts in thethree studied reproductive stages from each breed arepresented in Table 1 and in Figure 1. The mean lengthsof oviduct varied in pre-laying stage (F3,16=31.49,
P<0.05) obtained from 6 months indigenous(22.63±7.5cm), 3 months sonali (9.53±1.05cm) and 4
months RIR (13.53±0.73cm) chickens. In laying stage
the mean lengths were increased and achieved highest
values comparing to pre-laying and regressing stages inthree experimental breeds. Indigenous laying hens at 8
months showed mean length 50.76±2.17cm, sonali at 6
months 73.16±6.40cm and RIR at 10 months
91.26±4.23cm
werestatisticallysignificant(F3,16=12.29, P<0.05). After laying stage adult hens
started to terminate egg production for physiologicaldysfunction and thus getting regressed. The meanvalues of oviduct length for regressing hens decreased
from the laying stage and they showed significant
difference (F3,16=11.06, P<0.05) among indigenous(44.93±9.37cm) at 10 months, sonali (53.76±5.67cm)
at 19 months and RIR (83.93±2.48cm) breed at 18
months.
The mean weights (gm) of the oviducts in the three
reproductive stages from three breeds are presented inTable 1 and in Figure 2. The total oviduct mean
weights varied in pre-laying stage (F3,16=43.52,
P<0.05) in experimental different breeds (4.56±1.55gm
for indigenous at 6 months, 2.77±0.63gm for sonali at
3 months and 3.70±1.90gm for RIR at 4 months). Dueto accumulation of secretory substances in the cells forthe egg production, the oviduct weights were rapidly
increased in all three breeds and they showedsignificant variations among them (F3,16=7.44, P<0.05).
The laying hen oviduct mean weight for indigenous at
8 months was 32.30±1.94gm, for sonali at 6 months
was 51.53±7.52gm, and for RIR at 10 months was
93.26±6.39gm. Due to regression, the hens showeddecreased oviduct average weights which were53

Proceedings of International Symposium on Animal Production and Conservation for Sustainable Development 2018
SESSION 2 - Avian Production and Reproduction -: 03

13.53±0.73cm and mean weight 3.70±1.90gm of RIR
chicken at pre-laying stage were not in agreement withthe findings of different workers.

Length of oviduct (cm)

Indigenous

Sonalicommercial hybrid Dekalb strain oviduct weight atlaying (14months) 51gm (Yu et al., 1972). All thesefindings of cross bred layers were not comparable withthe present results with mean length and weight values
73.16±6.40cm and 51.53±7.52gm for 6 months. The
RIR layer showed the mean length at 5-6 months was
68.50±0.39cm (Naragude et al., 1999), laying stage
after 5 months the length was 67.74cm and weight 77.2
gm (Singh, 2001). The total length of oviduct at 10
months was 68 cm (Rahman, 2013), at 13 months was
63.26 ±2.51cm and weight was 41.64±7.08 gm (Shyam,2007), ISA brown chicken oviduct mean length was
73.45±0.96cm at 9 months (Rahman et al., 1999),
laying turkey had mean length 72.8±5.3cm (Parto et al.,2011). Very recently, Preedaa et al. (2018) reported themean length of WH Layer at 11 months was
71.85±0.56 cm and weight was 52.50±1.24gm. The
oviduct during laying stage increased rapidly withhighest values (91.26±4.23cm and 93.26±6.39gm at 10
months) and thus the present findings of RIR at laying
stage were superior to others reports.

RIR

100
90
80
70
60
50
40
30
20
10
0
Pre-laying
Laying Regressing
Reproductive stages of breeds

Indigenous
Fig. 1 Comparison of total oviduct length (cm)from pre-laying, laying and regressing stages ofindigenous, sonali and RIR chicken breeds.

Sonali

RIR

Weight of oviduct (gm)

100
90
80
70
60
50
40
30
20
10
0

There are many reports on oviductal morphologicalmeasurements (length and weight) of adult laying hensof indigenous, exotic RIR and a few on cross bredsonali chickens. Indigenous adult laying hens (Gallus
gallus dometicus) between 12-18 months had mean
length 71.85±5.45cm and mean weight 56.15±9.67gm(Mohammadpour et al., 2012), 8-11 months
46.18±9.49cm and 24.45±1.81gm (Mishra et al., 2014)
and only mean length at 9 months was 69.02 ± 0.93cm(Rahman et al., 1999), at 8 months 45.0cm length and23.60gm weight in native guinea fowl (Abdul-Rahman
et al., 2018). The entire oviduct was weighed
19.77±1.48gm for normal feathered, 19.8±3.50gm for
Naked Neck and 25.33±5.52gm for Frizzled Featheredin three Nigerian indigenous adult laying chickens(Mahmud et al., 2017) and indigenous adult turkey
hens (Meleagris gallopavo) at 11-14 months had mean
length 87.57±37cm and weight 59.66±1.52gm (Mirhish
and Nsaif, 2013). From the current study it was evident
that the mean length and weight from indigenous
chicken 50.76±2.17cm 32.30±1.94gm at 8 months wascompatible with the reports except Mohammadpour etal. (2012) for more age, Mahmud et al. (2017) for
different genotypes and Mirhish and Nsaif (2013) for
different species.

Pre-laying
LayingRegressing
Reproductive stages of breeds
Fig. 2 Comparison of total oviduct weight (gm)from pre-laying, laying and regressing stages ofindigenous, sonali and RIR chicken breeds.
The commercial hybrid Dekalb strain oviduct weight at
regressing stage 4.6 gm (19 months) (Yu et al., 1972),
14-16 months showed 38.20±4.20gm total oviductweight with a regressing rate (Yu et al., 1973). The
reduction of oviduct occurred during induced molting
in WH hens (Sudaresan et al., 2006) and in laying
Hylinebrown
females(64.98ௗ±ௗ1.91 g
to41.05ௗ±ௗ2.76 g at 13 months) (LeĞniak-Walentyn andHrabia, 2016). The total weight of oviduct varied withstage of ovarian development (F5,54=31.9, P<0.001) i.e.
oviduct weight regressed from its laying stage of its
follicular absence in a passerine bird (Williams andAmes, 2004). Khokhlov (2008) reported that dynamic
increase occurred in infundibulum of Lohman Brown
hens in laying stage and decreased in regressing stage.
The present results of regressing stages (indigenous

The crossbred Cairo L-2 strain matured sexually at 5
months and oviduct weight was 43.1±1.27 gm (Nassar
et al., 2017). F2 generation from a cross between White
Leghorn (WL) and Dongxiang Blue-shelled (DX) layer
chickens displayed the oviduct length 48.18±6.70cm
and weight 41.46±9.69gm (Shen et al., 2017). The

54

Proceedings of International Symposium on Animal Production and Conservation for Sustainable Development 2018
SESSION 2 - Avian Production and Reproduction -: 03

44.93±9.37cm, 19.83±7.65gm; sonali 53.76±5.67cm,
27.90±13.64gm; RIR 83.93±2.48cm, 59.30±2.44gm)
from three breeds were comparable with theabovementioned findings.
The breeds of present study are used by egg producers
and breeders for dual purposes i.e. egg and meatproduction and of them the sonali and RIR are rearedcommercially where the eggs of sonali are used only inhatcheries for meat production. In pre-laying stage,
immature chickens do not produce egg and theiroviduct development with increased length and weightis associated with differentiation and specialization ofcells from the particular segments of oviduct for the
production of egg-specific proteins in laying stage. The
molting or regressing process leads to dynamicchanges in morphology, physiology and function of the
reproductive tract of laying hens (Jeong et al., 2013)
and thus egg laying got terminated. The present results
explored the fact that the increased morphometricvalues of oviduct have been found in laying stages inall breeds comparing to that of pre-laying andregressing reproductive stages. The oviducts from RIR
hens had largest length and heaviest weight of oviductduring laying and then regressed in regressing stages
and the indigenous had highest values only in prelaying stage (Table 1, Figure 1 & 2). Considering all,the present findings made clear comparison among
three reproductive stages in chicken breeds on the basisof oviduct morphometry and also provided ahierarchical order as RIR‫ޓ‬sonali‫ޓ‬indigenous. Thechanges of morphological parameters of oviductrelated to the egg production efficiency in the studied
breeds and we are eagerly waiting for the investigation
to be conducted in the next.

Reference
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Conclusion
The mean lengths and weights of the entire oviduct ofindigenous, sonali and RIR hen breeds at pre-laying,laying and regressing stages were significantly
different (P<0.05). All the three breeds had similar
gross and morphometric patterns of oviduct increase
from the pre-laying to laying and decrease from laying
to regressing stages. So, this study in addition to its
contribution to the knowledge of comparative oviductal
morphology at different reproductive stages up todifferent breeds, has also established a baseline data forfurther comparative gross and morphometricreproductive studies in another breeds. A better
understanding of the morphological aspects of oviductwill provide the basis for improvements in theefficiency of chicken egg production and it will help todevelop improved technologies for the propagation ofendangered chicken breeds as well as other species ofpoultry.

Acknowledgement
This is a part of MS thesis work of Mst. Shamima
Khanam supervised by first author MAR in the
Department of Zoology, University of Rajshahi,
Bangladesh. Thanks to Chairman of the same
Department for providing facilities to conduct the
research work. The authors would like to thank poultry
growers and sellers for providing chickens and theirage information.

55

Proceedings of International Symposium on Animal Production and Conservation for Sustainable Development 2018
SESSION 2 - Avian Production and Reproduction -: 03

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Danmaigoro A Abdulsalam W Maaji M and
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Purushothaman MR and Murali N (2018) Effect of
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26) Rahman MA Baoyindeliger Iwasawa A andYoshizaki N (2007) Mechanism of chalaza formationin quail eggs. Cell and Tissue Research, 330:535-543.
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N (2009) VMO-II mediates the binding of thechalaziferous layer with the vitelline membrane inquail eggs. Journal of Poultry Science, 46: 240-248.
28) Rahman MM Baqui MA and Howlider MAR(2004) Egg production performance of RIR x Fayoumi
and Fayoumi x RIR crossbreed chicken under intensive

management in Bangladesh. Livestock Research for
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F (1997) Exotic hens under semi-scavenging conditionin Bangladesh. Livestock Research for Rural
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in the eggs, nestlings and adults of the barn swallow.
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Wang X Li Y Wang K Yang N (2017) A genome-wide
study to identify genes responsible for oviductdevelopment in chickens. PLoS ONE 12(12):e0189955.
32) Shyam KL (2007) Studies on grosshistomorphology and histochemistry of female
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34) Solomon S (2002) The oviduct in chaos. World’s
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35) Sundaresan NR Anish D Sastry KV Saxena VK
Mohan J Ahmed KA(2006) Cytokines in
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Proceedings of International Symposium on Animal Production and Conservation for Sustainable Development 2018
SESSION 3 - Animal Physiology and Production -: 01

Effect of total mixed ration ensiled with fermented juice of epiphytic lactic acid bacteria
on fermentation quality and energy expenditure
Yuli Yanti1,2 and Masato Yayota3,4
1
The United Graduate School of Agricultural Science, Gifu University, Japan2
Department of Animal Science, Faculty of Agriculture, Universitas Sebelas Maret, Jalan Ir Sutami
No. 36A Kentingan Surakarta, Jawa Tengah, Indonesia
3

4

Faculty of Applied Biological Sciences, Gifu University, Japan
Education and Research Center for Food Animal Health, Gifu University (GeFAH), JapanSUMMARY
This aim of this study was to determine the effect of total mixed ration (TMR) ensiled with fermented juice of epiphytic
lactic acid bacteria (FJLB) on fermentation quality and energy balance in ewes. The TMR were prepared from rice straw,
corn stover, brewer grain, tofu waste, steam flaked maize and mineral-vitamin. The treatments were: CON (no silage
additive added), FJLB, COM (commercial additive) and MIX (FJLB+COM). TMR was fermented for minimum 2
months. Four Suffolk ewes with the rumen canullas (7.5 ± 2.2 years old; 51.0 ± 19.5 kg) were assigned into 4 × 4 Latin
square design. The TMR was offered at the amount of 2% of BW on dry matter (DM) basis All the parameters observedin fermentation quality were not affected by silage additives (P>0.05), except for acetic acid concentration (P<0.01).
The COM and MIX treatment has higher acetic acid concentration than CON treatment (P<0.01). The chemicalcomposition of TMR silage was not affected by the silage additives (P>0.05). However, the DM content in FJLB
treatment tended to be higher than that in COM treatment (P=0.06). The silage additives did not show clear effect(P>0.05) on the gross energy (GE) intake, digestible energy (DE), energy digestibility, methane (CH4) energy andmetabolizable energy (ME). The TMR silage prepared from agriculture by-products with FJLB has good fermentation
quality and no negative effect on methane emission and energy utilization..
Keywords: energy utilization, fermentation quality, FJLB, methane emission, TMR silage

Introduction

ration (TMR). Food by product usually in wet type

Agricultural by-product has been known as feed fortherefore easy to deteriorate. Ensilaging the TMR is one

livestock in developing countries where the cropof technology to preserve the nutrient content of TMR.

production, such as rice paddy and maize, are abundant

An additive could be added to silage to improve thein amount. However, the utilization of these crop

fermentation quality. Fermented juice of epiphytic lactic

by-products is challenged by its low nutrient content;acid bacteria (FJLB) is a potential silage additive that has

mainly structural carbohydrates that will lead to low inbeen proved to increase the fermentation quality ofdigestibility then finally result in low animal production.

forage silage.

Further, in the process of fermentation in the rumen,

In our previous study, the TMR silage with FJLB

2-12% of ingested gross energy is converted to methane,contained similar amount of fiber to non-additive

which leads to reducing the efficiency of feedstuff

treatment. However, when FJLB treatment combined

utilization.

structural

with commercial silage additive, the fiber content ofcarbohydrates produces more methane emission in the

TMR silage was improved (Yanti and Yayota, 2017). It

rumen

may suggest that TMR silages with FJLB might

than

Feed
feed

that

contains

contained

many
low

non-structural

mitigates the methane emission and improve energy

carbohydrates (Archimède et al. 2011).

utilization of agricultural by-products.

To supply the balanced nutrient to livestock,agricultural by-product could be combined with other

Therefore, we hypothesized that FJLB treatment on

feed source, such as food by-product, into total mixed

TMR silage that prepared from agriculture by-product57

Proceedings of International Symposium on Animal Production and Conservation for Sustainable Development 2018
SESSION 3 - Animal Physiology and Production -: 01

will lead to reduce methane emission and more efficientacetic acid concentration (Table 1). The COM and MIX

energy utilization in ruminants. The objective of thistreatment has higher acetic acid concentration than CON

study was to determine the effect of TMR ensiled withtreatment (P<0.01). The chemical composition of TMRFJLB on fermentation quality and energy balance insilage was not affected by the silage additives (P>0.05).

ewes.

However, the DM content in FJLB treatment tended toMaterials and Methods

be higher than that in COM treatment (P=0.06). The

The proportion of material in TMR was 25, 23, 19.5, 19,silage additives did not show clear effect (P>0.05) on the12, 1.5% for rice straw, corn stover, brewer grain, tofu

GE intake, DE, energy digestibility, CH4 energy and ME

waste,(Table 2).

steam

flakedmaize

andmineral-vitamin,

Discussion

respectively. TMR was formulated to meet or exceed thenutrient requirement of sheep according to NRC (2007).

The Fleig point in all silages was over 97.99,

All the materials were mixed manually and added silage

indicating that all TMR silages had very good quality.

additive according to the following treatments. The

Wet type of food by-products including tofu waste andtreatments were: CON (no silage additive added), FJLB,

brewer grain contained lactic acid bacteria (LAB: 9 ×

COM (commercial additive: “Si-Master AC”®, Snow

107 and 6.4 × 107 cfu/g, respectively. Tanaka et al. 2001).

Brand Seed Co.,Ltd., Sapporo, Japan), and MIX

Thus, it would be sufficient to produce lactic acid and(FJLB+COM). The FJLB were prepared from Italianlowering pH to achieve good fermentation quality

ryegrass modified by Burenook et al. (2016). FJLB was

because well preserved silage will be obtained whensprayed 1% of fresh matter. The commercial additive

LAB level reaches 105 cfu/g (Cao et al. 2016).

dilutedandsprayedaccordingtothefactory

Acetic acid content in COM and MIX treatment wererecommendation. TMR were packed into polyethylene

higher than that in CON treatment. It can be explained

bag doubled with flexible container bag (capacity 100

that LAB in COM (Lactobacillus paracasei) waskg). Silages were made in four replications for eachheterofermentative type that can convert lactic acid into

treatment.

acetic acid. The heterofermentative LAB may also

Four Suffolk ewes with the rumen canullas have beenpresent in FJLB, because acetic acid in FJLB treatment

used (7.5 ± 2.2 years old; 51.0 ± 19.5 kg), then they weretended to be higher than CON treatment and becoming

assigned into 4 × 4 Latin square design. The ewes weregreater when combined with commercial additive (MIX).

housed in a metabolic cage individually. The TMR was

Other parameters in silage fermentation quality did notoffered at the amount of 2% of BW on dry matter basis.

differ among the treatments. Again, this was because

Adaptation was 8 days followed by collection period for

non-additive treatment also contained sufficient LAB for

6 days in each period. Fecal and urine were collected andgetting favorable fermentation.

handled.

GE intake was similar in ewes fed TMR silage with

The chemical composition of TMR silage and feces

different silage additive. This result might be caused by

was analyzed by the AOAC methods (AOAC, 2007).

the similar palatability of all TMR silage due to similar

The volatile fatty acid (VFA) in TMR silage was assayedsilage quality. The DE was not different among theusing gas chromatography. Fleig point was calculated astreatments. This is because similar chemical composition

described by Denek and Can (2006).

in all TMR silages led to have a similar rumen

The gross energy (GE) concentration of the samples

degradation and nutrient absorption in the intestinal tract.

was determined with a bomb calorimeter (CA-4PJ,

Predicted energy losses as methane in this study was

Shimadzu Corp., Kyoto, Japan). Digestible energy (DE)0.08 of GE, and this value agreed with the value of

was computed from difference GE intake and fecal

predicted methane in Adesogan et al. (1998) that often

energy. Metabolizable energy (ME) was computed fromquoted for feeds at maintenance. This result implied that

DE minus the urinary energy (UE) and energy losses

TMR silage prepared from agriculture by-products doesfrom methane. Methane production was estimatednot have any adverse effect on methane emission.

according to Blaxter and Clapperton (1965).

In conclusion, TMR silage prepared from agriculture

Results

by-products with FJLB has good fermentation quality

All the parameters observed in fermentation quality wereand no negative effect on methane emission and energy

not affected by silage additives (P>0.05), except for

utilization.

58

Proceedings of International Symposium on Animal Production and Conservation for Sustainable Development 2018
SESSION 3 - Animal Physiology and Production -: 01

Acknowledgements

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irradiation of microflora and fermentation quality ofsilages containing tofu cake or brewer's grain.
Grassland Science 47: 274-282.
Yanti Y and Yayota M (2017) Effect of silages additiveon fermentation quality and nutritive value of total
mixed ration silage made from agricultural
by-products. Proceeding Grassland Science 63: 70.

The first author thanks to Directorate General of
Resources for Research, Technology and Higher
Education, Republic of Indonesia for granting doctoral
scholarship through Beasiswa Pendidikan Pascasarjana
Luar Negeri (BPLN 101.21/E4.4/2015).

References
Adesogan AT, Owen E, and Givens DI (1998) The
chemical composition, digestibility and energy valueof fermented and urea-treated whole crop wheat
harvested at three stages of maturity. Grass and Forage
Science 53:66-75.
Association of Official Analytical Chemist, AOAC
(2007) Official methods of analysis of the associationof official analytical chemist, 18th edition. Associationof official analytical chemists. Virginia, USA.
Archimède H, Eugène M, Magdeleine CM, Boval M,
Martin C, Morgavi DP, Lecomte P and Doreau M(2011) Comparison of methane production between
C3 and C4 grasses and legumes. Animal Feed Scienceand Technology 166-167: 59-64.
Blaxter KL and Clapperton JL (1965) Prediction of theammount of methane produced by ruminant, British
Journal of Nutrition 19: 511-522.
Bureenok S, Sisaath K, Yuangklang C, Vasupen K and
Schonewille JT (2016) Ensiling characteristics ofsilages of stylo legume (Stylosanthes guianensis),guinea grass (Panicum maximum) and their mixture,

Table 1. Fermentation quality and chemical composition of TMR ensilaged with fermented juice of epiphytic lactic
acid bacteria
Treatment

Item

P-value

CON

FJLB

COM

MIX

pH

4.58 ± 0.13

4.41 ± 0.13

4.25 ± 0.10

4.33 ± 0.10

0.303

Lactic acid (g/kg DM)

31.51 ± 3.40

37.43 ± 3.78

37.80 ± 4.36

40.15 ± 2

0.379

Acetic acid

13.22 ± 2.88b

16.72 ± 0.78ab

21.46 ± 1.59a

24.22 ± 1.21a

0.005

Propionic acid

0.89 ± 0.34

0.92 ± 0.27

0.96 ± 0.31

1.08 ± 0.36

0.976

Butyric acid0.46 ± 0.09

0.27 ± 0.10

0.42 ± 0.15

0.1 ± 0.06

0.125

97.99 ± 5.55

106.23 ± 4.86

109.35 ± 3.03

108.52 ± 3.94

0.301

DM

38.0 ± 0.33

38.8 ± 0.44

37.1 ± 0.45

38.4 ± 0.35

0.055

CP (%DM)

12.5 ± 0.16

12.9 ± 0.28

13.2 ± 0.20

13.1 ± 0.31

0.227

aNDFom (%DM)

48.9 ± 1.08

47.6 ± 1.41

47.7 ± 0.74

46.9 ± 0.34

0.581

Fermentation quality

VFA (g/kg DM)

Fleig point
Chemical composition

CON: control (no additive added), FJLB: fermented juice of epiphytic lactic acid bacteria additive, COM: Commercial silage additive, MIX:FJLB+COM, VFA: volatile fatty acid, CP: crude protein, aNDFom: Į-amylase neutral detergent fiber exclusive ash, DM: dry matter. The values withdifferent superscript within the same row are significantly different at 5% level. Values are mean ± standard error.

59

Proceedings of International Symposium on Animal Production and Conservation for Sustainable Development 2018
SESSION 3 - Animal Physiology and Production -: 01

Table 2. Effect of different silage additives in TMR silage prepared from agriculture by-product on energy utilization in
ewes
Parameters
DMI (g/kg BW

0.75

/day)

GEI (MJ/kg BW
DE (MJ/ kg BW

0.75

0.75

/day)

FJLB

COM

MIX

P-value48.3 ± 3.25

45.9 ± 3.37

39.5 ± 5.19

44.2 ± 1.90.404

0.9 ± 0.06

0.9 ± 0.06

0.8 ± 0.07

0.8 ± 0.04

0.415

0.6 ± 0.03

0.6 ± 0.03

0.5 ± 0.04

0.6 ± 0.05

0.663

CH4 energy (MJ/100 MJ GE)

8.5 ± 0.09

8.4 ± 0.11

8.4 ± 0.17

8.4 ± 0.19

0.898

0.75

0.5 ± 0.03

0.5 ± 0.02

0.4 ± 0.03

0.5 ± 0.05

0.490

ME (MJ/BW

/day)

CON

/day)

CON: control (no additive added), FJLB: fermented juice of epiphytic lactic acid bacteria additive, COM: Commercial
silage additive, MIX: FJLB+COM, DMI: dry matter intake; GEI: gross energy intake; DE: digestible energy; ME:
metabolizable energy.
Values are mean ± standard error.

60

Proceedings of International Symposium on Animal Production and Conservation for Sustainable Development 2018
SESSION 3 - Animal Physiology and Production -: 02

Potential use of inulin as a prebiotic to maintain gastrointestinal healthin monogastric animals
Adi Ratriyanto*, Eka Handayanta, Nuzul Widyas and Sigit Prastowo
Department of Animal Science, Faculty of Agriculture, Sebelas Maret University, Surakarta Indonesia*Corresponding E-mail: ratriyanto@staff.uns.ac.id

SUMMARY
Due to the ban of antibiotics in animal production, new concepts have been developed to secure animal health andperformance. Prebiotics are naturally occurring compound considered to beneficially affect the composition and activityof the microflora in the gastrointestinal tract (GIT), thus, may exert positive influences on immune response of theanimals. Inulin has attracted considerable attention as a non-digestible oligosaccharide with prebiotic properties andoffered as potential substitutes for in-feed antibiotics. It is a polydisperse carbohydrate consisting mainly of ȕ-(2,1)
fructosyl-fructan links and shows a degree of polymerization ranging from 20–60 fructose units. Inulin is nothydrolytically digested by mammalian enzymes in the upper GIT, but is selectively fermented in the lower tract andthus may support the growth of beneficial bacterial populations, such as Bifidobacteria and Lactobacilli. Microbial
fermentation results in the formation of short-chain fatty acids, mainly acetic, propionic, and butyric, as well as lactic
acid, resulting in a lower pH and prevention of enteric colonization of potentially harmful bacteria. Furthermore, thereis evidence that inulin addition to the diet improved crude ash, crude fiber and neutral detergent fiber digestibility,
which would be concomitant with the maintenance of a beneficial intestinal environment.

Introduction

polymerization (DP) ranging from 20–60 fructose units

The use of antibiotics as feed additive was banned firstly[5]. It is originated, for example, chicory roots

in Sweden in 1986, followed by some other countries(Cichorium intybus) and Jerusalem artichoke tubers

and the European Union in 2006 [1]. In Indonesia, the(Heliantus tuberosus) [2]. This review focuses on theuse of antibiotic growth promoters has been banned in

effect of inulin on performance traits, intestinal microbes2017. During the first years after restriction of in-feeds

and nutrient digestibility in monogastric animals.

antibiotics in Sweden, there were increase in the use
antibiotics as therapeutic [1]. Therefore, there is growing

Effects of inulin on animal performanceinterest on natural substances as substitute of in-feeds

The prebiotic function of inulin has been confirmed in

antibiotics to secure the animal health and performance,

studies with humans and animals [6,7]. Studies inincluding prebiotics [2].

humans suggest that inulin is indigestible in the upper

Prebiotics are oligosaccharides or polysaccharides thatpart of GIT [6], which was confirmed by studies in pigs

are not hydrolytically digested by mammalian enzymes[8]. In their observation, Yasuda et al. [8], found the lackin the upper gastrointestinal tract (GIT), but areof inulin degradation in the upper GIT of pigs. By

selectively fermented in the lower tract and thus may

contrast, results of other studies, however, revealed that

support the growth of beneficial bacterial populations,about 57% of inulin was already digested in the stomach

such as Bifidobacteria and Lactobacilli [3]. Certain

and small intestine of pigs [9]. The higher pre-cecal

dietary non-digestible oligosaccharides (NDO), such asdigestibility of inulin in pigs compared to humans could

inulin and oligofructose are considered as prebiotics [4].

be due to higher numbers of bacteria in the small

Inulin and other NDO are considered as potential

intestine of pigs [9].

substitutes for in-feed antibiotics due to their negative

Generally, inulin can be utilized by Bifidobacteria,

impact on the growth of harmful bacteria [3]. Inulin is a

Lactobacilli, Bacteroides, and Enterococci but cannot be

polydisperse carbohydrate consisting mainly of ȕ-(2,1)

utilized by E. coli [2,10]. It is fermented by anaerobic

fructosyl-fructan

bacteria to be utilized as energy source, thereby

links

and

shows

a

degreeof61

Proceedings of International Symposium on Animal Production and Conservation for Sustainable Development 2018
SESSION 3 - Animal Physiology and Production -: 02

providing carbon skeletons for their own growth [10].

Table 1. Effects of inulin on performance, immune systems and

The mechanisms through which beneficial bacteria may

blood metabolites

exert their inhibitory effects on potentially harmful

AnimalInulin Effects

Referencebacteria are partly related to the production of

Broiler
Broiler
Broiler

Ĺ performance and carcass traits
Ĺ performanceĹ feed intake
Ĺ intestinal immune function (IgA
concentration in cecum and mucin
mRNA expression in jejunum)Ĺ growth performanceĻ blood lipids (triglyceride and VLDL)Ĺ serum sodium levels, creatine kinase,creatinine
Ĺ number of genes, processes andpathways with putative involvement ingrowth and performanceĹ immune immune status
Ĺ weight gain, feed efficiency
No effects on performanceNo effects on ND antibody titerĹ weight gain
Ĺ weight gainĹ growth performanceNo effects on performance and carcass
traitsĹ egg production and quality
Ļ blood triglyceride and cholesterolĹ egg production and quality

Praveen et al. [13]Kareem et al. [14]Huang et al. [29]short-chain fatty acids (SCFA) and lactic acid during
fermentation, resulting in a lower pH and prevention ofenteric colonization of potentially harmful bacteria [4].
In addition, inulin is supposed to improve the absorption

Broilerof certain minerals in pigs [11] and poultry [12], and to
stimulate the synthesis of B-vitamins [4].

Broiler

Beneficial effects of inulin in maintaining intestinal
Broilerhealth is associated with improvement in animal
performance. Studies on the effects of inulin on animal
performance are presented in Table 1. Inclusion of inulin

Broiler
Broilerin the diet improved performance of broiler chickens[13,14], laying hens [15,16], pigs [17] and rabbits [18].
This improvement is associated with modification in

Broiler
Broiler
Broiler
Quailssubstances which involved in protein or energy
metabolism such as carnitine, creatine and albumin[18,19]. Moreover, Sevane et al. [20] observed that
inclusion of inulin in the diet improved number of genes,

Layinghens
Layinghens
Layinghens
Rabbits

processes and pathways with putative involvement ingrowth, performance and immune status of chickens. The
lactic acid-producing bacteria are considered to be
immunomodulatory which directly or indirectly affect
the GIT and systemic defense functions. Dietary inulinthat increase the density of these bacteria may enhance
defense mechanisms of the host and increase resistancePigs

to various health challenges [21].

Pigs

Effects of inulin on intestinal bacterial
populations and microbial metabolites
with inulin on intestinal bacterial populations (Table 2)effects

indicated inconsistent results in pigs [22,23], and poultry

Sevane et al. [20]Izadi et al. [31]Elrayeh et al. [32]Nabizadeh [33]Rebolé et al. [7]Awad et al. [34]
Majewska et al.
[35]
Sang-Oh et al.
[15]
Chen et al. [16]Chen et al. [12]Attia et al. [18]Grela et al. [17]SamoliĔska andGrela [11]on

microbial

populations,someauthorsconcluded that the prebiotic effects of inulin in pigs areas well [7,24]. Loh et al. [22] and Eberhard et al. [25] didless pronounced compared to those observed in humans,

not find a stimulation of Bifidobacteria and Lactobacilli

poultry and rodents [9,25,36]. However, it should bein the duodenum, jejunum, cecum and colon of pigs due

noted that the discrepancy between studies on inulin

to dietary inulin supplementation. By contrast, Lynch etdegradation and effects on microbial populations may be

al. [23] found an increase in the cecal Bifidobacteria

attributed, at least in part, to differences in the

populations in pigs, accompanied with a decreased E.

methodology and origin of the test substrates [8,36], thecoli population following inulin supplementation in theage of the animal and type of legumes and cereals in thediet. Similarly, inulin supplementation increased thediet [25]. As was pointed out by Henry and Saini [37],number of Bifidobacteria and Lactobacilli in broilers [7],legumes and cereals, such as soybean meal, lupines

laying hens [15] and turkeys [26], but inhibited E. coli

wheat, barley and, commonly used in feed formulationand Salmonella growth in the small intestine and ceca of

contain substantial amounts of NDO, which may have

broilers [26–28].
on inulin

Ates et al. [19]

Ĺ= increased, Ļ= decreased

Studies pertaining to effects of dietary supplementation

Based on the findings

Ĺ eggshell weight and strengthĹ ash, calcium, and phosphorus in tibia
Ļ mortality rate
Ĺ performanceĹ blood hemoglobin, albumin
Ĺ weight gain and feed efficiency
Ĺ immunoglobulin A and GĹ weight gainĹ plasma trace elements (Zn, Fe, Cu)
and hemoglobin

Taraz et al. [30]masked and diluted the effect of dietary inulin

degradation and its

62

Proceedings of International Symposium on Animal Production and Conservation for Sustainable Development 2018
SESSION 3 - Animal Physiology and Production -: 02

supplementation. The prebiotic efficiency of NDO

The addition of 2% inulin to a diet of growing pigs

depends also on the DP of the fructans. NDO showing a

improved ileal and total tract crude ash [9,36] and ileal

longer DP are more resistant to fermentation, thus

crude fiber digestibilities [9], indicating an enhanced

leading to fermentation more distally in the GIT [38].

fermentation of fiber in the small intestine of pigs.

Moreover, dietary NDO may not selectively increase

Furthermore, supplementation of inulin in the diet did

Bifidobacteria when the initial level of the indigenous

not affect total tract dry matter, organic matter, crude

population is already relatively high prior to dietary

protein, neutral detergent fiber and energy digestibilities

inclusion [22].

in pigs [43].
The improvement in nutrient digestibility due to NDOTable 2. Effects of inulin on microbial populations andsupplementation

metabolites in the gastrointestinal tract

maintenance of a beneficial intestinal environment [2,9].

AnimalInulin Effects

Furthermore,

Reference

Broiler
Broiler

Ļ Salmonella counts
Huges et al. [28]Kareem et al. [14]
Ĺ faecal lactic acid bacteria
Ļ Enterobacteriaceae counts
Ĺ faecal acetic acid, propionic acid,
SCFA
BroilerĹ Bifidobacteria counts
Nabizadeh [33]Ļ E. coli counts
Rebolé et al. [7]BroilerĹ Bifidobacteria and lactobacilli
countsĹ butyric and d-lactic acids
Rehman et al. [24]BroilerĹ acetic acidNo effects on intestinal bacterial
communities
BroilerĹ Bifidobacteria, Lactobacillus counts Xu et al. [27]Ļ E. coli counts
LocalĻ intestine pH
Krismiyanto et al.
chickens Ĺ Lactobacilli[39]Ļ E. coli
LayingĹ Bifidobacterium and Lactobacillus Sang-Oh et al.
[15]
hens
population
Ļ Escheria coli and Salmonella
TurkeyĹ Bifidobacteria, Lactobacillus counts Juskiewicz et al.
Ļ E. coli counts[26]
TurkeyĹ faecal microbial activity
Stanczuk et al.
Ĺ short-chain fatty acids
[40]
Pigs
Ĺ Bifidobacteria
Metzler et al. [41]Loh et al. [22]Pigs
No effect on Bifidobacteria and
Lactobacilli
Ĺ butyric acidĻ acetic acid and total SCFA
Pigs
No effect on Bifidobacteria andEberhard et al.
Lactobacilli
[25]
Pigs
Ĺ Bifidobacteria
Lynch et al. [23]Ļ E. coli

would

the

be

lack

concomitantofaneffect

with
of

the

inulinsupplementation in several studies may have been due toa masking or dilution effect of native NDO from cereals
and legumes used in these diets [44]. It is possible, thatminimal effect in nutrient digestibility is linked to alower intestinal transit time, a common attribute offermentable fiber [3]. In addition, it has been wellestablished that the extent of microbial fermentation andnutrient digestibility is partly dependent on the source
and level of NDO supplementation and transit time ofdigesta in the GIT [44].
Table 3. Effects of inulin on nutrient digestibility and intestinal
morphology
AnimalInulin Effects

Reference

Broiler
Broiler
Broiler
BroilerĹ jejunum histomorphometry
Izadi et al. [31]
Ĺ intestine and cecum length
Elrayeh et al. [32]
Ĺ intestinal morphology
Nabizadeh [33]
Ĺ crude protein and fat digestibility, Alzueta et al. [42]amino acids and fatty acids
Broiler
Ĺ intestinal morphology and nutrient Awad et al. [34]absorption
Laying hens Ĺ small and large intestine length
Chen et al. [16]Rabbits
Ĺ crude protein digestibility
Attia et al. [18]Pigs
Ĺ crude ash digestibility
Branner et al. [36]Pigs
Ĺ crude ash and fiber digestibility
Böhmer et al. [9]Pigs
No effects on nutrient digestibility
Pierce et al. [43]Ĺ= increased, Ļ= decreased

Conclusion
Inulin is potential to be applied in the diet of monogastric

Ĺ= increased, Ļ= decreased, SCFA= short-chain fatty acids

animals and has been shown to improve animal
performance including poultry, rabbits and pigs due to its

Effects of inulin on nutrient digestibility

mode of action in modification of intestinal microbes. It

Studies in poultry showed that inulin increased crude

cannot be enzymatically digested in the upper part ofprotein and fat digestibility, amino acids and fatty acids

gastrointestinal tract, but selectively fermented bydigestibilities [42] associated with modification in

beneficial

intestinal morphology [31,33] as presented in Table 3.

bacteria

suchasBifidobacteriaandLactobacilli. Increasing number of these bacteria

Different to poultry, studies in piglets revealed only

stimulate the production of short-chain fatty acids andminor effects of inulin on nutrient digestibility [9,36].

63

Proceedings of International Symposium on Animal Production and Conservation for Sustainable Development 2018
SESSION 3 - Animal Physiology and Production -: 02

lactic acid, lead to reduction in harmful bacteria andoverview J. Ren. Nutr. 12 76–86

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improvementin

intestinal

health.

Inanderythrocyte

indicesin

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blood properties Poult. Sci. J. 6604 151–8

additives or in combination on bacterial populations65Proceedings of International Symposium on Animal Production and Conservation for Sustainable Development 2018
SESSION 3 - Animal Physiology and Production -: 02

in the gastrointestinal tract of weaned pigs Arch.
Anim. Nutr. 63 427–41[42] Alzueta C, Rodríguez M L, Ortiz L T, Rebolé A andTreviño J 2010 Effects of inulin on growth
performance, nutrient digestibility and metabolisableenergy in broiler chickens Br. Poult. Sci. 51 393–8[43] Pierce K M, Callan J J, Mccarthy P and Doherty J V
O 2005 Performance of weanling pigs offered low
or high lactose diets supplemented with avilamycin
or inulin Anim. Sci. 80 313–8[44] Houdijk J G M, Hartemink R, Verstegen M W A andBosch M W 2002 Effects of dietary non-digestible
oligosaccharides on microbial characteristics of ileal
chyme and faeces in weaner pigs Arch. Anim. Nutr.
56 297–307

66

Proceedings of International Symposium on Animal Production and Conservation for Sustainable Development 2018
SESSION 3 - Animal Physiology and Production -: 03

Mechanisms of sperm-storage in the avian oviductżMei Matsuzaki and Tomohiro Sasanami
Faculty of Agriculture, Shizuoka University
SUMMARY
Although successful fertilization depends on timely encounters between sperm and egg, the decoupling ofmating and fertilization often confers reproductive advantages to internally fertilizing animals. In several vertebrategroups, which employ internal fertilization, post-copulatory sperm viability is prolonged by storage in specializedorgans within the female reproductive tract. In birds, ejaculated sperm can be stored in a quiescent state in motility
within oviductal sperm storage tubules (SSTs), thereby retaining fertilizability for up to 15 weeks at body temperature(41 °C); however, the mechanism by which motile sperm become quiescent within SSTs is currently unknown. Here,
we show that low oxygen and high lactic acid concentrations are established in quail SSTs, thus resulting sperm motility
inactivation in the SSTs. These findings suggested a novel physiological role of lactic acid for the sperm quiescence in
the SSTs and opened up a new opportunity for technological improvement in prolonging sperm longevity at ambient orbody temperature.

Introduction

fractions were tested for their activity of sperm motility

In internal fertilizers, successful fertilization depends oninactivation and the molecular structure of purified

the timely arrival of both sperm and egg at the site of

bioactive component was determined by NMR analysis.

fertilization. To achieve this, females of some of these

We examined the synthetic pathway of

species are capable of storing spermatozoa in theirbioactive component by incubating the isolated SST withreproductive tracts until their eggs are ready to beor without an inhibitor of glycolysis. We also addressed

fertilized. This phenomenon is common in many

the mechanism of how this component inhibits sperm

non-mammalianmotility by measuring sperm dynein ATPase activity andanimals,

includinginsects,fish,

amphibians, reptiles, and birds.

axoneme sliding in the presence of the active component.

Results and Discussions

In avian species, sperm storage tubules (SSTs),
simple tubular invaginations located between the vagina

We first identified sperm motility inactivating factor

and uterus, serve as sperm storage sites. Once ejaculated,from the UVJ extracts. The UVJ extracts were separated

spermatozoa migrate to and stay in the lumen of SSTs,

and tested inhibitory effects on sperm motility. As thewhere they can remain their fertilizability for longresult, lactic acid was identified as a sperm motility

periods of time (up to 15 weeks) at a normal bodyinactivation factor from the bioactive fraction. The lactic

temperature of 41 °C. Previous studies have explored theacid concentration in the SST cells was higher than thatphysiological roles of SSTs in sperm quiescenceof UVJ epithelial cells (Fig.1; Matsuzaki et al., 2015). In(Sasanami et al., 2013); however, the underlying

addition, the lactic acid production in the SSTs occurred

molecular mechanism of this process has remained ain the presence of glucose and significantly inhibited bymystery. In this study, we hypothesized that SSTs containthe addition of 2-deoxyglucose, an inhibitor of glycolytic

a substance(s) that renders sperm motility inactive.

enzyme (Fig.2; Matsuzaki et al., 2015). Therefore, it was

Based on this hypothesis, we aimed to identify thecomponent(s) responsible for sperm quiescence within
SSTs.

Material and Method
Excised quail UVJ tissues were minced in extraction
buffer and extracted for 3 h on ice. The UVJ extracts
wereseparatedbygel-filtration

chromatography,

high-performance liquid chromatography (HPLC), andpreparative thin-layer chromatography (PLC). The
Fig.1 Measurement of lactic acid in the SST cells.

67Proceedings of International Symposium on Animal Production and Conservation for Sustainable Development 2018
SESSION 3 - Animal Physiology and Production -: 03

suggested that SST cells are able to produce lactic acidmanner. Hence, it follows that the quiescence of sperm

via

performed

flagellar beating is related with decreasing dynein

histochemical analysis and it was revealed that theATPase activity induced by intracellular acidification. In

mitochondrial activity of SST epithelial cells was

fact, pHi of the resident sperm in the SSTs were

suppressed due to the limited oxygen supply around thedecreased approximately 6.3.

SST

Conclusion

glycolysis.

cells.

In

Subsequently,

addition,theweexpressionofmonocarboxylate transporter 4 (MCT4), which is

In this study, following conclusions were obtained.

responsible for lactic acid transport was evident in the(1) Lactic acid was identified as sperm inactivation

SSTs by RT-PCR analysis. Based on these observations,factor derived from the UVJ extracts.

it is suggested that lactic acid is synthesized in the SST(2) SST epithelial cells produced lactic acid from glucose

epithelial cells via glycolysis and might be transported to

via the glycolysis under hypoxic conditions, andthe lumen through the function of MCT4.

probably released into SST lumen via MCT4.

Second, we investigated the mechanism of(3) Sperm motility suppression during the storage insperm motility inhibition by lactic acid. Consequently, it

SSTs was caused by decreasing dynein ATPase activity

was observed that sperm motility was inhibited not only

resulted in intracellular acidification of sperm inby lactic acid, but also by other organic or nonorganic

response to low pH of SST lumen.

acids. According to the measurement of extra- andThese findings suggested that lactic acidintracellular pH of sperm (pHe and pHi) in the presence

produced in the SST cells reduces sperm motility duringof various organic acids, pHi was decreased in response

storage. However, we were unable to produce fertilized

to pHe decrease by the addition of any acids (Fig.3;

eggs by artificial insemination using in vitro stored

Matsuzaki et al., 2015). Furthermore, pHe and sperm

sperm, indicating that sperm preservation requires

motility had a strong positive correlation, suggesting that

unknown factor(s) other than lactic acid. Further studies

inactivation of the sperm motility is caused by

are needed to uncover the mechanism of sperm storage

decreasing luminal pHi due to the presence of highin avian species.

Acknowledgement

This work was supported by a Grant-in-Aid for Scientific
Research (B) (General) (24380153 to T.S.), a
Grant-in-Aid for Scientific Research in Innovative Areas(24112710 to T.S.), a Grant-in-Aid for Challenging
Exploratory Research (25660211 to T.S.), Japanese
Association for Marine Biology (JAMBIO) (No. 24–64
and No. 25–57 to T.S.), Toukai Foundation forTechnology (No. 7 to T.S.) and Japan Advanced Plant
Science Network.

References
Matsuzaki, M., Mizushima, S., Hiyama, G., Hirohashi,

Fig.2 Lactic acid production in isolated SSTs.

N., Shiba, K., Inaba, K., Suzuki, T., Dohra, H., Ohnishi,T., Sato, Y., Kohsaka, T., Ichikawa, Y., Atsumi, Y.,
Yoshimura, T., Sasanami, T. Lactic acid is a sperm
motility inactivation factor in the sperm storage tubules.
Scientific Reports, 5:17643 (2015).
Sasanami, T., Matsuzaki, M., Mizushima, S. & Hiyama,
G. Sperm storage in the female reproductive tract inbirds. Journal of Reproduction and Development, 59,
334–338 (2013).

Fig.3 Relation between extra- or intracellular pH
and lactic acid concentration.
concentration lactic acid. On the other hand, sperm
dynein ATPase activity as well as axoneme sliding of
de-membrane sperm decreased in a pH-dependent68

Proceedings of International Symposium on Animal Production and Conservation for Sustainable Development 2018
SESSION 4 - Animal Conservation -: 01

Threats and conservation of Sumatran primates
RizaldiDepartment of Biology, Faculty of Mathematics and Natural Sciences, Andalas University, Indonesia

SUMMARY
Sumatra is a rich biodiversity island laid on the tropical region of Indonesian territory. It is home to about 17
species of non-human primates including great apes (orangutans), gibbons, macaque monkeys, leaf monkeys, and slow
loris. All those primate species live in various habitat types range from natural forest, degraded forest to humanhabituated environments. Development of agricultural industries and other human facilities across the island have given
threats to all species at different intensity levels. The threat intensities depend on several factors; forest-dependent
species, landscape contour, diet composition and living in close contact with the human. Habitat loss and fragmentation
are the most serious threat to those primates although other threats are also alarming. Even though primates are very
important biodiversity richness in the island, I found fewer efforts have been made for protection and lack studies havebeen conducted to support conservation. Study on geographical distribution and population status of each species areurgently needed in order to suggest authorities to make appropriate conservation plans and to take immediate actions.
Here, I am presenting distribution surveys of primate populations conducted in the central region of Sumatra.
This surveys aimed to identify ongoing threats to each species in this region and to understand their current distribution
due to habitat alteration. This survey found tiny scattered populations of leaf monkeys, a moderate population of
macaques and very limited distribution of gibbons. Large oil palm plantation area could not be tolerated by any primate
species but scattered forest remnant and rubber gum forest remains in the plantation have compensated dietaryrequirements of leaf monkeys and macaques. I am also presenting a comparative study of agile gibbon- aforest-dependent species. This study compared between living in fragmented and non-fragmented forest habitat in orderto understand how forest fragmentation threats the gibbons. I analyzed demographic parameters, ranging behavior andfood resources of the gibbons. I have observed population increased of agile gibbons (Hylobates agilis agilis) living infragmented forest habitat although they have very limited home ranges compared to non-fragmented habitat. We will
discuss minimum habitat requirement for a sustainable population of the purely arboreal primates. These studies suggest
an immediate evaluation of the conservation status of Sumatran primates especially each species and sub-species of leafmonkeys. Recommendation for in-situ conservation, partnerships and applicable management conservation would be
discussed.

69

Proceedings of International Symposium on Animal Production and Conservation for Sustainable Development 2018
SESSION 4 - Animal Conservation -: 02

Ex situ conservation of Javan pangolin (Manis javanica) in Bogor Agricultural
University, Indonesia 
1,2
Chairun Nisa , Irmanida Batubara2,3㸪Burhanuddin Masyud2,4 and Entang Iskandar2,5
1

Faculty of Veterinary Medicine, 2Tropical Biopharmaca Research Center, 3Department of Chemistry

Faculty of Mathematics and Natural Sciences, 4Faculty of Forestry, 5Primate Research Center, Bogor
Agricultural University, Indonesia
SUMMARY
Javan pangolin is an unique mammals that belong to protected species according the Indonesian regulation, and listed as
critically endangered species in IUCN red list, and APPENDIX I of CITES. However, exploitation of the animals still
occurring massively that so conservation of this animal is strongly needed. The captive breeding is the best alternative
solution and legally justified for sustainable using. Bogor Agricultural University has concerned on conservation of theJavan pangolin since 2011 and has engaged maintenance in captivity started on 2014 till now. Firstly the study on
ecological aspect, such as population and habitat characteristic was done, and continued with development study ofpangolin captive breeding. The study started with adaptation of behavior and daily activity of pangolins in captive
breeding cage, along with study of feed formulation as a substitute for natural feed, and continued with study ofbreeding and reproduction technology and management. The results showed that Javan pangolins had a good
adaptability and were able to survive in captivity as an artificial habitat. A key factor in the life of the Javan pangolins in
captivity was determined by the successful provision of artificial feed formulas as an alternative to natural feed
substitution. Javan pangolins shown positive in pairing and social interaction as an early indicator of breeding andreproductive processes in captivity. Implementation of Javan pangolin breeding and reproduction technology incaptivity is still an important challenge in the captive process.

Introduction

Pangolin scales have long been used in TCM (traditional
Chinese medicine) and are believed to be a cure forJavan pangolin (Manis javanica) is one of Indonesia's

certain diseases, such as rheumatism, skin, fever, evenunique biodiversity assets. It is known as pangolin ortumors or cancer; whereas the meat is believed toscaly anteater because of its scaly and ant-eating bodyincrease vitality (Bräutigam et al. 1994). This has led to(Figure 1). In the classification of pangolins belongs tomassive exploitation of pangolins.

the order Pholidota which only has one family Manidae,
one genus Manis and eight species. M. javanica is one offour species distributed in Asia, while four other species
in Africa. Currently the pangolins belong to the protectedanimals under Law no. 5 of 1990 and PP No.7 of 1999,
as well as a list of red list or critically endangered species
by IUCN, although CITES still places it in the Appendix
II list.
Fig.1. Javan Pangolin with scaly body A. roll up
and B doing activity in the cageAt present, the exploitation of pangolins has reached an
alarming level, so that the population in nature is underserious threat that can lead to the extinction of theseIn the past ten years, anteaters are thought to be the mostanimals. Pangolin is widely exploited because somesmuggled animals from Indonesia, with the ultimate goalpeople, especially the Chinese people, believe inbeing mainly China. While conservation efforts both intreatment and healing using pangolin body parts.

situ and ex situ are still far from expectations. One of the70Proceedings of International Symposium on Animal Production and Conservation for Sustainable Development 2018
SESSION 4 - Animal Conservation -: 02

ex situ conservation efforts that has been carried out isbreeding, but the success rate is still low. This is mainly

Result and Discussions

due to the constraints of specific natural feeds, which are

Study on ecological aspect of Javan Pangolin:

ants and termites (expensive in prices and in-continuous

population and habitat characteristic (2011-2012).

supply), and still lacking data on reproductive biology

The research started on 2011 with the population andinformation. So, the aims of the study is to conserve

habitat characteristic in West Java, Central and South

Javan pangolin by captive breeding as the best

Kalimantan, and North Sumatera. The objective of thisalternative solution and legally justified for sustainable

study is to get an overview of population conditions andusing.

habitat characteristics as a basis or reference forconstructing and designing of artificial habitat inMaterial and Method

captivity.

Bogor Agricultural University started the Javan pangolin

At present the condition of pangolin populations inconservation on 2011 till now. The research scheme isnature is under serious threat. From the results ofshown in Fig 2. The first stage is study on ecologicalecological studies and pangolin populations in several

aspect of Javan Pangolin: population and habitat

regions of the three major islands in Indonesia, namely:

characteristic (2011-2012). The location for this study isWest Java, North Sumatra, South Kalimantan, andin a number of sites that become part of the area ofCentral Kalimantan, shows that the pangolin population

spread, namely: Java Island, especially West Java,in these locations is already very low at only 3-80

Kalimantan (Central Kalimantan and South Kalimantan)

pangolins, even in some rare locations (population < 3and Sumatra especially North Sumatra.

tails) (Masy'ud et al., 2012). Pangolin make nest hole in

The second stage is development study of pangolin

the ground or in the tree trunk. The habitat of Pangolin

captive breeding (2013-now). On this stage three stage ofand their nest in the nature is shown in Figure 3.

research are conducted, namelya.

The study of behavioral adaptation and daily
activity of pangolins in captive breeding cage.

b.

The study of feed food as a substitute fornatural foodc.

The study of breeding and reproduction
technology and management

Fig. 3 Pangolin habitat and nest
The illegal hunting of pangolin directly from nature
which is carried out continuously, uncontrolled illegal
trade and utilization without being followed by breeding
activity will result in the extinction of these animals. In
addition, the pangolin population decline is also thought
due to the conversion of functional areas of pangolin
habitat into fields or plantations, resulting in habitat
destruction and reduced availability of pangolin feed innature. Pangolins eat ants (ordo Hymenoptera), termites(ordo Isoptera) and red earth ants (Myrmicaria sp.) as themost preferential or preferable feed in their natural
habitat.

Fig. 2 Research Scheme

This animal is a solitary and nocturnal animal, so it only71

Proceedings of International Symposium on Animal Production and Conservation for Sustainable Development 2018
SESSION 4 - Animal Conservation -: 02

has afternoon to night activities (Novriyanti 2011). So far,in captivity, calculate feed consume and positive effect tosome zoos and conservation centers, such as Singapore

growth (FCR, feed conversion rate), body weight andZoo, Taipei Zoo Taiwan, and Carnivore and Pangolin

survivality of pangolin in captivity. Artificial feed

Conservation Project (CPCP) Vietnam, have beenformula is made by utilizing chicken egg and meat, meal

successful in keeping pangolin to produce puppies fromworm, cricket powder and other supplement, combine

marriage in captive, adapting feeding replacements fromwith ant egg (local: kroto). The study conducted this year

natural feed which is relatively expensive and difficult toto adapt pangolins with an artificial feed formula showed

maintain continuity of availability.

good results. Feed formulations that were suitable andsufficient for the metabolic needs of the pangolin body.

Development Study of Pangolin Captive Breeding

Our research team has successfully adapted pangolins(2013-now)
Captive breeding is one of the ex situ conservationthat were kept in captivity with artificial feed formula
substitutes (Nisa' et al. 2015).

efforts, although the level of success is still low. So far,the information about the successful maintenance of
pangolins in captivity by providing substitute feed
formulas has only been reported by several zoos abroad,such as the Singapore Zoo and Taipei Zoo.
TropBRC has 4 pangolins in the captive breeding; 2 isfemale and 2 is male (Figure 4). Each pangolin wasmaintaining individually, that in each cage facilitatedwith connecting door. Generally, pangolin was shown
well adaptive in artificial habitat (cage). The pangolin in
captive breeding was doing rest in the wooden box or in
the logs as the enrichment of the cage, eating, moving in
the ground of cage, and doing social interaction between
male and female Pangolin (Figure 5).

Fig. 5 Pangolin in captivity: a. rest in wooden
block, b. rest in logs, c. feeding activity, d. walking
in the ground, e. social interaction between female
and maleMorphological information of female reproductive
organs of pangolins (M. javanica) (Rachmawati, 2011)and males (Akmal, 2014) have been reported. These
things are expected to become a database for the successof pangolin breeding in Indonesia. So, for the next study
will conduct is related to the reproductive behavior of
pangolin in captivity, supported by analyses of hormone
profile and steroid metabolite of female reproductive
hormone of pangolins. In an effort to provide greater

Fig. 4 Pangolin in captivity: A. female-1, B.
male-1, C. female-2, D. male-2

space for a pair of pangolins to be mated, the connecting
door between the two cages is opened. For hormone
analyses will be carried out non-invasively, using fecal

Since the natural food for Pangolin is difficult to find, theand urine samples as well as invasively using blood

supply is not continuous, and the price is also expensive,

samples. The results of this study are expected as an

we performed the study on artificial feed formulation for

initial effort to breed pangolins in captivity to support ex

substitution the natural feed. This study is started on

situ conservation.

2015. The formula was adopted from Singapore Zoo, but

Information of female reproductive biology such as themodified using local feed. The objective of the step is tocharacteristics of the ovary and estrous cycle as well as

create or find the optimum artificial feed formula tomonitoring methods are very important because thereplace or decrease the use of natural food for pangolins

72Proceedings of International Symposium on Animal Production and Conservation for Sustainable Development 2018
SESSION 4 - Animal Conservation -: 02

success of animal breeding depends on female fertility(Agil, 2007). One method of monitoring the estrous

Referencecycle is through the profile of the reproductive hormone,

Agil, M. 2007. Reproductive Biology of the Sumatranespecially estrogen and progesterone. Pangolin is an

Rhinoceros Dicerorhinus sumatrensis (Fisher 1814).

animal that easily stressed, so the monitoring method

Dissertasion. Bogor: Graduate School of Bogor
Agricultural University.

should be avoid the animal stress, such noninvasive
method. The profile of hormone can be determined using

Akmal Y. 2014. Anatomy of the male reproductive organ

noninvasive method from fecal and urine sample, as wellof pangolin (Manis javanica). Acta Vet 2 (2): 75-81
Bräutigam A, J Howes, T Humphreys, and J Hutton. 1994.

invasive method using blood sample.

Recent information on the status and utilization of

Reproductive hormones are steroid hormones which after

African pangolin. Traffic Bull.15: 15-22.

metabolic processes, especially in the liver, their
metabolites will be excreted through feces and urine.

Masy’ud B, E Iskandar, C Nisa’, ED Purwakusumah, A

Excretion of reproductive metabolites varies in shape andManshur. 2012. Study of ecology and population ofamount in each species. Generally reproductive hormone

pangolin (Manis Javanica Desmarest, 1822): Case

metabolites are excreted through feces and urine in the

study on West Java, North Sumatera, South

form of oestron, oestradiol, pregnanolon or pregnanediol.

Kalimantan,

In some species hormone metabolites are excreted

Biofarmaca Research Center, Institute for Researchmostly through feces, while some other species throughand Community Services, Bogor Agriculturalurine. Although hormonal profile analyses of several

University.

andcentralKalimantan.

Bogor:

animal species has been studied, careful use of thisNisa’ C. 2015. Feed formulation for substituting natural

noninvasive method carried out in new species mustfeed of pangolin (Manis javanica) to support ex situ

really be the main concern. Characterization of types ofconservation. Research Report. Bogor: Institute forreproductive hormone metabolites and hormone assay

Research

validation that will be used must be done carefully when

Agricultural University.

andCommunity

Services,Bogormeasuring hormone levels of new species, so that the

Novriyanti. 2011. Study on breeding management, feed

results are accurate and can be used to examine

consumption and palatability, and daily activity of

reproductive problems of the species (Agil, 2007).

pangolin (Manis Javanica Desmarest, 1822) at UD
Multi Jaya Abadi North Sumatera. Thesis. Facultyof Forestry, Bogor Agricultural University.

Conclusion

Rachmawati

Javan Pangolins have a good adaptability and are able to

AF.

2011.

Morphology

offemalesurvive in captivity as an artificial habitat. One of the key

reproductive organ of javan pangolin (Manis

factors in the life of the Javan pangolins in captivity isjavanica) with special references to the follicle

determined by the successful provision of artificial feeddevelopment and distribution of carbohydrate in theformulas as an alternative to natural food substitution.

ovarium. Thesis. Faculty of Veterinary Medicine,Javan Pangolins shown positive pairing and social

Bogor Agricultural University.

interaction as an early indicator of breeding andreproductive processes in captivity. The implementationof javan pangolin breeding and reproduction technology
in captivity is still an important challenge in the captive
process.

Acknowledgement
The authors thanks to PT Primax for the financial
support on the first stage of this research and to the
Ministry of Research Technology and Higher Education
Republic of Indonesia for research funding on theschemeHigher

Education

Excellence

Research(Penelitian Unggulan Perguruan Tinggi).

73

Proceedings of International Symposium on Animal Production and Conservation for Sustainable Development 2018
SESSION 4 - Animal Conservation -: 03

Molecular Analysis of Tor spp. (Cyprinidae) in Sumatra
Dewi Imelda Roesma, Djong Hon Tjong, Warnety Munir, Dyta Rabbani Aidil
Biologi Department, Faculty of Mathematics and Natural Sciences, Andalas University

SUMMARY
The molecular study among Tor spp. in Sumatra (T. douronensis, T. tambra, T. tambroides and T. soro) has been
conducted using partial sequence of Cytochrome b (Cyt b) and Cytochrome Oxidase I (COI) genes. DNA amplification
was done using PCR machine. Analysis was done on 53 sequences of Cyt b (588 bp) and 61 sequences of COI gene(572 bp).

Sequence divergences among Tor spp. in Sumatra were less than 7% and 3.5% for Cytb and COI

respectively. While sequence divergences between Tor spp. in Sumatra and Tor spp. in other region in Asia were lessthan 7.3% and 4.5% for Cytb and COI respectively. The low of sequence divergences of Tor spp. do not support the
separated them into four different species.

Introduction

variation of T. tambroides in Batang Tarusan river

Tor (Cyprinidae) is the genus of freshwater fish which(Wibowo et al., 2013; Wibowo and Kaban, 2015), and thecommonly known as Mahseer or Semah group,

genetic variation of T. douronensis in West Sumatra

widespread in the Trans Himalaya region (Thailand,(Roesma et al., 2017). Although Tor in Indonesia,

Nepal, India and Myanmar) and Southeast Asia (Thailand,

especially in Sumatra, became one of the market

Cambodia, Vietnam, South China, Malay Peninsula andcommodities, the cultivation of Tor has not been done inIndonesia) (Kottelat et al., 1993, Rainboth, 1996, Kottelat,well.

2013). Tor lives in a clean and fast flowing water river

threats could have a future impact on Tor population

habitat inland and also found in cold water in highstability in nature, while, the study of Tor, especially inaltitudes area and warm water such as India (T. putitota

their genetic diversity, has not been finished. The breeder

and T. tor) (Roberts, 1999; Bhatt et al., 2004). Tor species

reported that the cultivation business was not satisfactory

became one of the fishes with high economic value andbecause of lack of support for genetic data. De Silva etbecame the favorite in the fishing game or ornamental fishal., (2004) concluded that the genetic information is(Ng, 2004).

necessary for the succes of the cultivate efforts.

The overfishing, habitat degradation or other

Currently, Tor comprised of 10 species (Kottelat et

Currently, molecular studies have been conducted inal., 2013) which previously consisted of 20 species (Kiat,

Tor groups using gene markers on mitochondrial DNA

2001). There are still many conflicting in the classified(cytochrome b and cytochrome oxidase I) because theyof Tor species, raising doubts on the clarity of Tor

have conserved regions, evolutionary rate is faster and aretaxonomy. Tor is distinguished from other Cyprinidae

able to categorize at species level (Esa et al., 2006;

groups based on the presence of the lobe (median lobe)

Nadiatul et al., 2011; Wibowo and Kaban, 2015). Basedat the bottom of the mouth (Zhou and Chu, 1996). In

on the previuos studies, an intensive study of Tor spp. inIndonesia, (Haryono and Tjakrawidjaya, 2006) reported

Sumatra using molecular analysis (cytochrome b andfrom the Zoology Museum Bogoriensis there are four

cytochrome oxidase I gene) is necessary to establish a

species namely T. soro, T. douronensis, T. tambra, and T.

clear taxonomy, to know the closeness of the relationshiptambroides. According to Kottelat et al. (1993), the fourto the success strategy of the crosses in cultivation andspecies are distinguished only based on the presence andappropriate

size of the median lobe, there are no other characteristics

geographical and environmental conditions.

conservation

efforts

considering

thethat clearly distinguish among the species.

Material and Method

The studies of Tor in Indonesia is limited tomorphological studies (Haryono and Tjakrawidjaya,
2006), gonad development (Haryono, 2006), genetic

Samples collection
A number of 28 individuals of Tor spp. were collectedstructure of T. tambroides in South Sumatra and genetic

from 13 populations (25 individuals) in West Sumatra, 274

Proceedings of International Symposium on Animal Production and Conservation for Sustainable Development 2018
SESSION 4 - Animal Conservation -: 03

populations (3 individuals) in Jambi for Cyt b and 15

Result and Discussions

populations (19 individuals) in West Sumatera, (2Molecular Analysis:

individuals) in Jambi and 4 populations (7 individuals) in
North Sumatera for COI analysis. A piece of the tissue

Cyt b Gene
A 588 bp from 740 bp which was successfully amplified

samples stored into Eppendorf tubes (1.5 ml) whichwere analyzed for 53 individual Cyt b gene sequences

containing 96% ethanol PA. Individual samples were

where 25 of them were obtained from the Genbank,stored within 10% formalin for a while and replaced with

NCBI.

70% ethanol for long term storage

was A: 29.60%, C: 30.20%, T: 27.70% and G: 12.50%.

DNA Isolation, PCR and DNA Sequencing

There were 241 bp (40.99%) variable site and 347 bp

The average of total nucleotide composition

DNA isolation was performed on the protocol Kit(59.01%) as a conserved site of a total of 588 bp without

INVITROGEN PureLinkTM Genomic DNA Mini Kit.

insertion or deletion. In the total of 241 variable sites,

The result of DNA isolation was used for amplification

165 bp (82.57%) as parsimony site and 42 bp (17.43%)

using PCR method with thermal cycler (SensoQuest).

as singleton site. In a total, there were 327 mutations

Primary used were forward 5’ CGA TTC TTY GCN TTC

among all sequences with the transition is more common

CAY TTC YT 3’ and 5’ CCT CCR ATC TTC CGA TTA

than transversion (Ts : Tv) with bias value R: 3.998.

CAA GAC 3’ (reverse) for Cyt b and forward (Fish F1: 5

Particularly to Tor spp. in Sumatra, 60 substitutions were

'TCAACCAACCACAAAGACATTGGCAC3')

observed

reverse

(Fish

and

consistingof

50

transitions

and

ten

transversions without insertion and deletion.

R1:5'TAGACTTCTGGGTGGCCA
The PCR product were

Construction of a phylogenetic tree using fourchecked in 2% agarose at a voltage of 100 V, power 20

methods (ML/NJ/ME/MP) in order to know theW for 50 min. Good quality PCR product were used as

relationship between Tor spp. which represented by NJ

samples for sequencing in MacroGen USA DNA

tree was shown in Fig. 1. NJ tree show similar topologies

Sequencing Laboratory.

for ML, ME and MP with a confidence level

Data Analysis
A total of 61 sequences of COI gene were analyzed

bootstraping 1000 times using the MEGA Version 6.0

consisting of 28 Tor spp. sequences in Sumatra and 33

using four methods, three main clusters were obtained,

sequences taken from Genbank NCBI included out groupsupported by highly bootstrap value 87/88/84/88 forspecies (Table not shown). DNA sequences (forward and

ME/NJ/ML/MP,

reverse) were assembled and edited using the DNA

showed the monophyletic relationship between Tor spp.

STAR program (Burland, 2000).DNA sequence of Tor

in Asia with Cobitis elongatoides and Cobitis taenia as

spp. in Sumatra compared with the sequences contained

outgroup.

AAGAATCA3') for COI gene.

program. Based on the analysis of the phylogenetic tree

respectively.

Phylogenetic

analysisin NCBI, to see the similarity of all sequences in

The first cluster consists of four species of Tor in

http://blast.ncbi.nlm. nih.gov/Blast.cgi. All sequences

Sumatra (first subcluster and second subcluster) andwere aligned using the Clustal X program (Thompson,

three species of Tor from China (third subcluster). The

1997). Sequences that have been align were edited using

second cluster consists three species of Tor in Sumatra

BIOEDIT program (Hall, 1999). The number ofand T. tambroides from Genbank (first subcluster), and T.

polymorphism sequences of the nucleotide sequences

putitora, T. khudree, and T. tor India (second subcluster).

was analyzed using the DNA Sequence Polymorphism

The third cluster consists of T. qiaojiensis and T.

5.10 (Rozas, 2003). Sequence divergences of allhemispinus and the genus which are said as a synonyms

sequences was analyzed using the Kimura-2 parameterof Tor, included Neolissocheilus, Labeobarbus, andmodel (K2P) in the MEGA 6.0 program (Tamura, 2013).

Barbus. The sequence divergences were calculated using

The phylogenetic tree was reconstructed based on four

Kimura 2 parameter method in MEGA Version 6.0

methods; Neighbors Joining (NJ), Maximum Evolution

program. Overall, Tor spp. in Asia has a sequence(ME), Maximum Likelihood (ML) and Maximum

divergences 0.0%-7.9% and Tor spp. in Sumatra with

Parsimony (MP) using MEGA 6.0 (Tamura, 2013).

sequence divergences 0.0%-6.9%.

75

Proceedings of International Symposium on Animal Production and Conservation for Sustainable Development 2018
SESSION 4 - Animal Conservation -: 03

According to Kartavtsev (2011), in the vertebrate,
the value of distance between sequence of Cyt b gene

COI Gene
Part of the COI gene was analyzed from 61of the species in the same genus

is 10.69 + 1.34.

individuals (33 of them from the Genbank, NCBI) using

Based on that value, all the species in the first cluster andforward (FISH F1) and reverse (FISH R1) primers. As

first subcluster in the second cluster should be consist ofsuch as 572 bp of COI gene sequences were analyzed

only one same species.

from the total length 720 bp which was successfully
amplified.
The average of total nucleotide composition was A:
26.40%, C: 27.60%, T: 29.60% and G: 16.40%. The
quantity of A + T (56.00%) is higher than G + C(44.00%) which is characteristic for the genome of thevertebrate mitochondrial as well as reported in other
Cyprinidae groups (Esa et al., 2012). There were 178 bp(31.12%) with the variable site and 394 bp (68.88%)
with the conserved site of a total of 572 bp without
insertion or deletion. In the total of 178 variable sites,
165 bp (92.17%) as parsimony site and 13 bp (7.83%) as
singleton site. In a total, there were 247 mutations among
all sequences with the transition is more common thantransversion (Ts : Tv) with bias value R: 5.584.
Particularly to Tor spp. in Sumatra, 31 substitutions wereobserved with 28 transitions and three transversions
without insertion and deletion.
Construction of phylogenetic tree using four methods(ML/NJ/ME/MP ( Fig. 2.). The NJ tree show similar
topologies to ML, ME and MP with a confidence levelbootstraping 1000 times using the MEGA Version 6.0
program. Based on analysis of the phylogenetic treeusing four methods, three main clusters were obtained,supported by highly bootstrap value 98/98/96/97 for
ME/NJ/ML/MP,

respectively.

Phylogenetic

analysis

showed monophyletic relationship between Tor spp. in
Asia and the out-group (Cobitis elongatoides and Cobitis
taenia).

Fig. 1 The Phylogenetic Tree of Tor spp. based on Cyt
b gene using bootstrap 1000 (ME/NJ/ML/MP)76

Proceedings of International Symposium on Animal Production and Conservation for Sustainable Development 2018
SESSION 4 - Animal Conservation -: 03

second cluster consists of three species of Tor in Sumatra,
T. putitora, T. khudree and T. tor India (first subcluster)and other Tor species from Asia (second subcluster).
While the third cluster consists of the genus which aresaid as a synonyms of Tor, included Neolissocheilus,
Labeobarbus, Barbus and Cyprinus.

The sequence

divergences is calculated using Kimura 2 parameter
method in MEGA Version 6.0 program. Overall, Tor spp.
in Asia has a sequence divergences 2.5%-3.5% and Tor
spp. in Sumatra with sequence divergences 0.0%-1.4%.
Again, according to Kartavtsev (2011), in the vertebrate,
the value of distance between sequences of CO1 gene of
the species in the same genus is 9.66 + 0.72. Based onthat value, all the species in the first cluster and firstsubcluster in the second cluster should be consist of only
one same species.
The result of the present study using Cyt b and
COI gene showed the overlapping placement of all the
species of Tor in Sumatra.

Nguyen et al. (2008)

reported that the unresolved relationship between Tor spp.
especially on T. douronensis with other species which
formed from three distinct lineages.

Diversity study of

T. tambroides and T. douronensis in Malaysia (Nguyen etal., 2006), showed that poorly resolved on T. tambroides
and T. douronensis taxonomy status with share the some
haplotype that allegedly due to gen flow, morphological
identification mistake or hybridization between species.
As previously described, these four species of
Tor were hard to be distinguished morphologically, thegrouping into different species generally based onpresence and the size of the median lobe (Kottelat et al.,1993). Controversial taxonomy status of Tor group had
occur since long time ago (Rainboth, 1996; Robert andKottelat, 1993). The result of present study do notsupport the separation of four species (T. douronensis, T.
tambroides, T. tambra and T. soro). The result of present
study is expected to be a reference for the researcher to
conduct the future studies for example in the breeding
management of Tor species and the determination ofconservation status.

Conclusion

Fig. 2 The Phylogenetic Tree of Tor spp.
based on COI gene using bootstrap 1000

The molecular analysis of present study using Cyt b and(ME/NJ/ML/MP)

COI gene do not support the separation of four species (T.
douronensis, T. tambroides, T. tambra and T. soro).

The first Cluster consists of four species of Tor in
Sumatra (first, second, and third subcluster with

Acknowledgement

sequence divergences 0.7%-1.4% between them).

This work was supported by Grants of Ministry of

The

77

Proceedings of International Symposium on Animal Production and Conservation for Sustainable Development 2018
SESSION 4 - Animal Conservation -: 03

Research, Technology and Higher Education to Andalas

andUniversity(No.06/UN.16.17/PP.UPT/LPPM/2017), thank

Mitochondrial DNA. 3, 55–65.

you very much for that chance.

We wish to express ourinanimals.

Selangor12) Kottelat, M. (2013). The Fishes of the Inland

1) Bhatt, J. P., Nautiyal, P. and Singh, H. R. (2004).
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71-91

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14) Nadiatul, H. H., Daud, S.K., Siraj, S.S., Sungan, S.,

Chornelia, A. (2017). Genetic Diversity of Tor

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De Silva, S.S, Ingram,B., Sungan, S.,Tinggi, D.,

oxidase I gene sequences. Iranian Journal ofGooley G., and S Y, Sim, S.Y.. (2004). Artificial

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propagation of the indigenous Tor species, empurau

15) Ng, C.K. (2004). Kings of the Rivers: Mahseer in(T. tambroides) and semah (T. douronensis),
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Malaysia and the Region. Inter Sea Fishery (M) Pte

Aquaculture Asia IX(4)Ltd. Selangor

5) Esa, Y. B., Siraj, S. S., Daud, S. K., Rahim, K. A. A.

16) Nguyen, T. T. T., Ingram, B. A., Sungan, S.,

N., Abdullah, M. T., Japning, J. R. R., and Tan, S. G.

Gooley, G., Sim, S.Y., Tinggi, D., and De Silva,(2006). Mitochondrial DNA diversity of Tor

S.S. (2006). Mitochondrial DNA diversity of

douronensis Valenciennes (Cyprinidae) in Malaysianbroodstock of two indigenous mahseer species, Tor

Borneo. Pertanika Journal of Tropical Agriculture

tambroides

Science. 29, 47-55.
6) Esa, Y. B., Siraj, S. S., Daud, S. K., Rahim, K. A.
A., Japning, J. R. R.

andbiogeography

Mekong FAO species Identification

Symposium Series. 41, 95-98.
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Field

Guide

f o Fishery Purpose. Rome : FoodandAgriculture Orgazation.1-265

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19) Roberst, T. R. (1999). Fishes of the Cyprinids genus

Improvement of Tambra Fish ((Tor spp: Cyprinidae)
from Indonesia.

(Pisces:18) Rainboth, W. J. (1996). Fishes of the Cambodian

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Studyspecies

Evolution. 48, 1223–1231

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79-PART 2UGSAS-GU & BWEL JOINT POSTER SESSIONON AGRICULTURAL            
AND BASIN WATER ENVIRONMENTAL SCIENCES

2018
POSTER PRESENTATIONSORGANIZERS:
THE UNITED GRADUATE SCHOOL OF AGRICULTURAL SCIENCE,
GIFU UNIVERSITY&
GIFU UNIVERSITY REARING PROGRAM
FOR BASIN WATER ENVIRONMENTAL LEADERS

Proceedings of International Symposium on Animal Production and Conservation for Sustainable Development 2018
UGSAS-GU & BWEL Joint Poster Session on Agricultural and Basin Water Environmental Sciences 2018: P01

Stemflow generation and dissolved organic matter properties in elated
to tree size and rainfall characteristic in a subtropical evergreen
broadleaved forest of central Japan
Siyu Chen 1, Ruoming Cao 1, Shinpei Yoshitake 2 and Toshiyuki Ohtsuka 3,
1. The United Graduate School of Agricultural Science, Gifu University, 1-1 Yanagido, Gifu 501-1193, Japan
2. Takayama Experimental Field Station, River Basin Research Center, Gifu University, 919-47 Takayama, Gifu 506-0815,
Japan
3. River Basin Research Center, Gifu University, 1-1 Yanagido, Gifu 501-1193, Japangeneration efficiency (stemflow volume, stemflow initiationthreshold, stemflow percentage and FR) at individual tree
scale, bigger trees would be more effective at generating
stemflow; (3) DOM properties of stemflow may be related totree size.

INTRODUCTION
Stemflow variability has been linked to meteorologicaland biological factors. The meteorological factors include
rainfall amount, intensity and duration; wind speed anddirection during rainfall events (Levia et al., 2011; Pyker etal., 2011; Van Stan et al., 2011). Linear relationship between
stemflow volume and rainfall amount has been reported bymany researchers (Price and Carlyle-Moses, 2003; Yang et al.,2008; Van Stan and Levia, 2010). However, the effects ofrainfall intensity on stemflow volume remain unclear. Somestudies showed that stemflow volume decreased with therainfall intensity and indicating that heavy rainfall may
overload stemflow pathways through the canopy (Crockford
and Richardson, 2000; Carlyle-Moses and Price, 2006;
Staelens et al., 2008), while some studies found that stemflow
increased with rainfall intensity (Van Stan et al., 2014, Zhang
et al., 2015). However, the reverse results may due to the great
difference of the maximum rainfall intensity in these studies,for example, the maximum rainfall intensity was up to
72mm/h in the research of Crockford and Richardson (2000),
while the maximum rainfall intensity was only 4.1mm/h in the
study of Van Stan et al., (2014). The biological factors include
canopy morphology, diameter at breast height (DBH), basal
area, bark morphology and branch angle, etc (Van Stan et al.,2011; Van Stan et al., 2014; Zhang et al., 2015). Previous
studies have shown that the bark morphology plays a crucialrole in stemflow volume and is a key constraint at smaller
rainfall magnitudes, bark water storage capacities differedsignificantly among different species (Levia and Herwitz,
2005; Carlyle-Moses and Price, 2006; Van Stan and Levia
2010). And forest canopies had a strong impact on theredistribution of rainfall and ecohydrological processes
occurring in forested landscapes (Owen et al., 2006).
Stemflow volume has been investigated to bary greatly as afunction of tree species. For example, coniferous trees weremore inclined to generate stemflow than broadleaved trees(Huber and Iroume, 2001); stemflow production of deciduoustrees varies over time as a result of foliation and defoliation(Staelens et al., 2008)
Therefore, the present study investigated stemflow
production of different tree sizes in more than 100 individual
rainfall events during 2 years (2016-2017) in a natural
Castanopsis cuspidata evergreen forest with different tree
sizes (evergreen broadleaved forest) of central Japan,emphasized on the stemflow of individual tree at individual
rainfall event scale. Accordingly, the novelty and main
objective of this study is to analyze the tree size associateswith rainfall characteristics effects on stemflow generation
basing on the large data base at individual rainfall event scale.
We hypothesized that: (1) rainfall amount and intensity havedifferent influences on stemflow volume, stemflow initiationthreshold,stemflow percentage and funneling ratio (FR); (2)tree size is an important factor to control the stemflow

MATERIALS AND METHODS
The study site is located on Mt. Kinka, central Japan. A 0.7
ha study plot (70 m  100 m) was established on the lower
slopes of Mt. Kinka (ca. 60 m a.s.l., 35r26͛ N, 136r47͛
E) in 1989. The study area has a subtropical monsoon climate
and the study forest is an evergreen broadleaved forest, which
has a basal area of 46.1 m2 ha-1, the dominant tree species ofthis forest is Castanopsis cuspidata, which accounted for
87.86% of basal area and is also the dominant canopy tree
species in the study plot (Chen et. al, 2017). The annual mean
temperature is 16.1 °C, and the mean temperature in thecoldest month, January, and the hottest month, August, are
4.4 °C, and 28.0 °C, respectively. The study area has abundantprecipitation, with an annual average precipitation of 1926
mm during 2016-2017. The climatic data was collected at aweather station near the study plot.
To measure stemflow hydrologic flux, 12 individual trees ofdominant species (C. cuspidata) were selected for observation,
including 4 DBH classes (20-30 cm, 30-40 cm, 40-50 cm, >50cm) with 3 individual trees in each DBH class. Stemflow
volumes were collected from June 2016 to December 2017
and represented 111 rainfall events. Stemflow samples of 12trees were collected from the sample reservoir tank twice amonth for chemical analysis.
RESULTS AND DISCUSSION.
Results indicated that individual stemflow volume of C.
cuspidata had an exponential relationship with incident
rainfall depth, stemflow volume increased with increasing
rainfall depth and followed a positive exponential function (p< 0.001), which was more significant in smaller (20 cm <DBH < 30 cm) trees. In general, individual stemflow volume
was increasing when rainfall intensity was less than 15 mm/h,
however, when rainfall intensity was greater than 15 mm/h,
stemflow tended to decrease with increasing rainfall intensity.
Moreover, stemflow volume increased with increasing
DBH class, DBH > 50 cm class produced the highest amountof stemflow (average = 14.87 L), more than twice of 20-30 cm
DBH’s (average = 35.65 L) (Fig. 1a). Further analysis
revealed that stemflow volume of different DBH classdemonstrated great differences under rainfall events withdifferent rainfall intensity. Significant differences were foundamong different DBH class during the rainfall events with
rainfall intensity was less than 15 mm/h, which were the larger
trees produced more stemflow than the smaller trees, the82Proceedings of International Symposium on Animal Production and Conservation for Sustainable Development 2018
UGSAS-GU & BWEL Joint Poster Session on Agricultural and Basin Water Environmental Sciences 2018: P01

average stemflow volume of DBH class > 50cm (35.8 ± 3.1 L)
was nearly 3 times as the 20-30 cm’(13.2 ± 1.2 L). Whereas
the average stemflow volume was 32.9 ± 9.8, 42.0 ± 9.9, 36.2
± 9.1 and 38.6 ± 10.0 for 20-30 cm, 30-40 cm, 40-50 cm andDBH >50 cm, respectively during the rainfall events with therainfall intensity was more than 15 mm/h, the differencesamong different DBH class were not significant.
Rainfall depth and intensity had a greater impact on
stemflow percentage of lager trees (DBH > 40 cm) than that ofsmall trees (DBH < 40 cm), and stemflow percentage of DBH> 40 cm trees decline sharply with increasing rainfall up to50mm (Fig.1b). Moreover, a negative exponential relationshipwas observed between stemflow percentage of incident
rainfall and rainfall intensity, Stemflow percentage decreased
with increasing rainfall intensity, and the stemflow percentage
decreased rapidly when rainfall intensity less than 15 mm/h,then the stemflow percentage tended to be stabilized no morethan 5%. Comparing the stemflow percentage in different
DBH class, we found significant differences between different
DBH class, stemflow percentage of 20 – 30 cm DBH class
was least (average = 3.8 ± 0.15%), while the DBH > 50 cm
DBH was largest (average = 10.6 ± 0.64%), stemflow
percentages of 30 – 40 cm and 40 – 50 cm DBH class was 6.4± 0.27 % and 9.0 ± 0.51%, respectively. Moreover, thevariabilities of stemflow percentages were different under
different rainfall intensity, the difference was significant
among different DBH class when rainfall intensity was lessthan 15mm/h, whereas the rainfall intensity was more than
15mm/h, the stemflow percentage appeared to be unaffected
tree sizes.
Similar to stemflow percentage, negative exponential
relationships were found between FR and incident rainfall
depth / rainfall intensity across all DBH class, and thecorrelations were more clear when tree size more than 40cm.
In contrast to stemflow percentage, the FR of C. cuspidata
was highest in 20 – 30 cm DBH class with an average of 17.9,
and lowest in DBH > 50 cm class with an average of 9.0. For
30 – 40 cm and 40 – 50 cm DBH class, FR averaged 15.8 and
11.6, respectively (Fig. 1c). The variabilities of FR among tree
sizes also different during different rainfall intensity, while therainfall intensity was less than 15mm/h, significant difference
was not found among the smaller trees ( DBH < 40 cm), but
found in the bigger trees ( DBH < 40 cm). However, whilerainfall intensity was more than 15mm/h, significant
difference was found in smaller trees (DBH< 40cm), and no
significant difference in bigger trees (DBH > 40cm).

Fig. 1: Box plots depict (a)stemflow volume, (b)Stemflow
percentage, (c) Funneling Ratio(FR) of different DBH classfor C. cuspidata (p<0.05).

REFERENCES
Carlyle-Moses, D.E., Price, A.G., 2006. Growing-season
stemflow production within a deciduous forest of southern
Ontario. Hydrol. Process. 20, 3651–3663.
Levia, D.F., Herwitz, S.R., 2005. Interspecific variation of
bark water storage capacity of three deciduous tree species inrelation to stemflow yield and solute flux to forest soils.
CATENA 64, 117–137.
Levia, D.F., Keim, R.F., Carlyle-Moses, D.E., Frost, E.E.,
2011. Throughfall and stemflow in wooded ecosystems. In:
Levia DF, Carlyle-Moses DE, Tanaka T (eds) Foresthydrology and biogeochemistry: synthesis of past research andfuture directions (Ecological Studies Series 216). Springer,
Heidelberg, pp 425–443.
Pypker, T.G., Levia, D.F., Staelens, J., Van Stan, J.T., 2011.
Canopy structure in relation to hydrological andbiogeochemical fluxes. In: Levia D.F., Carlyle-Moses D.E.,
Tanaka T (eds) Forest hydrology and biogeochemistry:
synthesis of past research and future directions. Springer,
Heidelberg, pp 371–388.
Staelens J., de Schrijver A., Verheyen K., Verhoest N.E.C.,
2008. Rainfall partitioning into throughfall, stemflow, andinterception within a single beech (Fagus sylvatica L.) canopy:
influence of foliation, rain event characteristics, andmeteorology. Hydrol Process 22:33–45
Van Stan, J.T., Levia, D.F., 2010. Inter- and intraspecificvariation of stemflow production from Fagus grandifolia Ehrh.
(American beech) and Liriodendron tulipifera L. (yellow
poplar) in relation to bark microrelief in the eastern UnitedStates. Ecohydrology 3:11–19.

83

Proceedings of International Symposium on Animal Production and Conservation for Sustainable Development 2018
UGSAS-GU & BWEL Joint Poster Session on Agricultural and Basin Water Environmental Sciences 2018: P02

Future changes of intensity and structure for landfalling typhoons in Japanby pseudo-global warming experiments
Masaya Toyoda, Jun Yoshino, and Tomonao Kobayashi
Environmental and Renewable Energy Systems Division, Graduate School of Engineering, Gifu University(2) High-resolution typhoon model (HTM)
The high-resolution typhoon model (hereafter HTM) is used tosimulate the intensity and size changes of overall life of
typhoons. HTM is based on the mesoscale meteorologicalmodel MM5 (Dudhia, 1993). The automatic movable nesting
technique and the several kinds of physical parameterizations(e.g. the ocean mixed layer, dissipative heating, and sea-sprayprocesses) are introduced into MM5 to express realistic
typhoon intensity and structure accurately (Yoshino et al.,2013). The triply nested computational domains used in thisstudy have a horizontal grid spacing of 27-km (D1), 9-km (D2),
and 3-km (D3), respectively. The movable nesting technique isapplied to both D2 and D3.

INTRODUCTION
Japan is a typhoon-prone country where typhoons constantly
approach with an average of 11.5 in a year and make landfall
with an average of 2.9 typhoons in a year. The IPCC Fifth
Assessment Report (IPCC, 2013) suggests future changes in thecharacteristics of tropical cyclones may occur, while there is asignificant amount of uncertainties in their predictions. In orderto estimate future change of coastal disasters such as storm
surge and high wave, it is important to project the intensity andstructure of future-climate typhoons. Although there are several
researches on the future changes for intensity and track offuture typhoons based on the statistical approaches (Tsuboki etal., 2014; Toyoda et al., 2017), there are few reports
investigating intensity and size of future typhoons based on the
dynamic approaches.
In this study, for a total of 52 typhoon which made landfall inJapan islands from 2000 to 2017, ensemble pseudo-global
warming experiments (CMIP5 RCP8.5 scenario: 2080 - 2099)
are conducted using a high-resolution typhoon model. Based on
the experimental database, we investigate the future change ofthe maximum wind speed (MW) and the radius of maximum
wind speed (RMW) at landfall time in Japan.

(3) Present climate and future climate experiments
In this study, first of all, present climate experiments arecarried out using NCEP FNL data as initial, boundary andassimilation
conditions.
We
conduct
future-climate
experiments by applying the method of pseudo-global warming
experiment (Sato et al., 2007) to estimate future changes ofintensity and structure of typhoons in comparison with present
climate observations. The input conditions for the pseudoglobal warming experiment are created by adding monthly
average global warming difference (GWDs) to NCEP FNL.
The GWDs are the difference of monthly mean fields between
the future climate present climate. Temperature, Sea surface
temperature, geopotential height, east-west wind speed, northsouth wind speed and relative humidity are used as the 3D
monthly mean fields derived from the general circulation
model GCMs. In this study, the monthly mean fields in the
future climate are averaged during 2080 - 2099 by a total of 8
GCMs of the CMIP5 RCP8.5 scenario (high-level reference
scenario: supposed not to do policy mitigation policy) In
addition, the monthly mean fields in the present climate are
averaged during 2000-2019 by 15 GCMs of the CMIP5
historical scenario (2000-2005) and 8 GCMs of the CMIP5
RCP8.5 scenario (2006-2019).

COMPUTATIONAL METHOD(1) Target typhoons
As target typhoon in this study, a total of 52 typhoons from2000 to 2017 which made landfall in the Japan islands are
selected. Fig. 1 shows the tracks of all 52 typhoons provided by
the Japan Meteorological Agency (JMA). According to the
JMA record, 53 typhoons made landfall in Japan during 2000
to 2017. Only one case, that is Typhoon CHATAAN (2002), is
excluded from the target typhoons because it is difficult to
calculate the whole life of the typhoon due to the deficiencies
of input initial and boundary conditions.

RESULTS AND DISCCUSION(1) Present climate experiments
First, we discuss the results of the MW and RMW of 52
typhoons simulated by the present climate experiments. The
MW and RMW simulated by HTM are smoothed in 6 hours.
Figure 2 compares the observed and simulated MW of 52
typhoons at the landfall time. Figure 3 also compares theobserved and simulated RMW of 52 landfalling typhoons. In
terms of MW, the bias error is +1.27 m/s, the root means squareerror is +5.35m/s, and the correlation coefficient is 0.75. In
terms of RMW, the bias error is +10.0 km, the root means
square error is +29.6 km, and the correlation coefficient is 0.73.
Therefore, it can be said that the results of the simulated global
warming experiments in the next section is highly reliable.
(2) Pseudo-global warming experiments

Fig.1 The track of 52 landing typhoons in 2000 ~2017 (JMA Best Track) covered in this research

84Proceedings of International Symposium on Animal Production and Conservation for Sustainable Development 2018
UGSAS-GU & BWEL Joint Poster Session on Agricultural and Basin Water Environmental Sciences 2018: P02

Table.1 Summarize of results of pseudoglobal warming experiments (Boldnumber mean significant results).

CONCLUSION
In this study, we conducted present climate experiments andpseudo-global warming experiments for 52 landfalling
typhoons in Japan from 2000 to 2017. It became clear that
under the future climate, the maximum wind speed of typhoon
is increased, on the other hands, the radius of maximum windspeed tends to be decreased by the future global warming. Thechanges in the characteristics of landfalling typhoons in Japanare considerable in the future. It is expected that future trendsof typhoon intensity and structure obtained in this research canbe utilized for disaster prevention and disaster reduction
measures in the future.

Fig.2 A scatter diagram and statistical scores of the MW
between estimation by CNTRL and observation
by JMA.

REFERENCES
IPCC: Climate Change 2013 -The Physical Science Ba-sis-,Cambridge University Press, 1535p., 2013.
Japan Meteorological Agency (JMA) 㸸 Typhoon statistics
http://www.data.jma.go.jp/fcd/yoho/typhoon/statistics/index.
html
Japan Meteorological Agency (JMA)㸸Typhoon location table
http://www.data.jma.go.jp/fcd/ yoho/typhoon/index.html
Dudhia, J., A nonhydrostatic version of the Penn State-NCAR
mesoscale model: Validation test and simulation of an
Atlantic cyclone and cold front, Mon. Wea. Rev., Vol.121,
pp.1493-1513, (1993).
Sato, T., F. Kijima and A. Kitoh: Projection of global warming
onto regional precipitation over Mongolia using a regional
climate model, Journal of Hydrology, Vol.333, pp.144-154,
2007.
Tsuboki, K., M. Yoshioka, T. Shinoda, M. Kato, S. Kanada andA. Kitoh: Future increase of supertyphoon intensity
associated with climate change, Geophysical Research
Letters, Vol.42, pp.646-652, 2014.
Toyoda, M., J. Yoshino and T. Kobayashi: Comparison of
future change and its uncertainty on typhoon intensity
between typhoon Haiyan (2013) and typhoon Melor (2009),
JSCE B2 (Coastal Engineering), Vol.73, No.2, pp. I_217I_222 (in Japanese), 2017.
Yoshino,J., S. Arakawa, S. Shimada and T. Kobayashi:
Intensity predictions on the 2004 pacific typhoons using 2-D
and 3-D Typhoon models, JSCE B2 (Coastal Engineering),
Vol.69, No.2, pp.I_1256-I_1260 (in Japanese), 2013.
Yoshino, J., S. Arakawa, M. Toyoda and T. Kobayashi: Intercomparison of global warming scenarios for typhoon
intensity change using a High-Resolution Typhoon Model,
JSCE B2 (Coastal Engineering), Vol.71, No.2, pp. I_1519I_1524 (in Japanese), 2015.

Fig.3 A scatter diagram and statistical scores of the
MW between estimation by CNTRL andobservation by JMA.
Next, we compare with pseudo-global warming experiments
present climate experiments and discuss future changes in MW
and RMW at typhoon landfall time. (Table.1). The average
MW and the average RMW at landfall time are 32.9 m/s and102.5 km in the present climate and are 38.4 m / s and 90.2 km
in the future climate, respectively. The future changes are +5.48 m/s and -12.3 km, and there are significant differences in
one-tailed test at a significance level of 5%. 41 typhoons showthat the MW are increased, and the greatest change between the
future climate and present climate is +22.5 m/s by Typhoon
USAGI (2007). In addition, 14 landfalling typhoons
corresponding to about 27% of the total typhoons are
categorized as the strength of either "very strong" or "furious"
under the future climate. On the other hands, 32 typhoons
indicate that the RMW is decreased, and the largest futurechange is - Km by Typhoon PABUK (2011). Therefore, future
landfalling typhoons in Japan tend to increase the MW and to
decrease the RMW. It is expected that the future trends oftyphoon intensity and structure provided by this study will be
useful to establish and redevelop the policy of typhoon-related
disaster mitigation and prevention.

85

Proceedings of International Symposium on Animal Production and Conservation for Sustainable Development 2018
UGSAS-GU & BWEL Joint Poster Session on Agricultural and Basin Water Environmental Sciences 2018: P03

Historical of agriculture transition and livelihood change in northern Laosafter road construction: Case study in Sone District, Houaphan Province
Cahyo Wisnu Rubiyanto1 and Isao Hirota2
1. The United Graduate School of Agricultural Science, Gifu University, Japan
2. Faculty of Applied Biological Sciences, Gifu University, JapanINTRODUCTIONRESULTS

Rural area has characterized as low population size, low
educational level, low socioeconomic status, high rates ofchronic disease and mortality, and low access to health careservices (Sharkey and Horel 2008). In Laos, 80% of
population live in countryside (UNDP 2004), where they have
low standards of infrastructure, especially roads. Road
construction is one of important factors altering rural
livelihoods. Improving road access is considered to be aneffective way of improving livelihoods and reducing rural
poverty (Rigg 2006; Warr 2006).
Local people in Laos have heavily depended on natural
resources and now the livelihood system is about to change.
The impacts of road construction bring various kinds ofchanges of local society and nature. The expansion ofagricultural field and following destruction of habitats ofvarious natural resources might be observed at local level on
one hand, and it can bring opportunity to access more cash
income source and contribute decrease poverty on the otherhand. To know the dynamics of local livelihood and natural
resource from various aspects is indispensable to present
sustainable system and relationship between society andnature.
This study compares two villages with different histories
and conditions of road; (1) Houay Su village (middle access)and (2) Bong village (bad access), in Sone district, Houaphan
province, northern Laos after preliminary survey of 14 villages
in the district and examine how road construction influence
the dynamics of natural resources and rural livelihood. This
study aims to clarify the historical rural livelihood change byfocusing on commercialization of agricultural products andnon-timber forest products (NTFPs), economic improvement
at village and household levels, historical transition ofagricultural system such as expansion of agricultural land andfollowing deforestation of surrounding environment between
two villages..

Livelihood changes in both villages are shown in Fig. 1 andFig. 2 Fig. 1 shows that the villagers in Bong village havecultivated rice in upland field. Number of household collecting
NTFPs have increased and red mushroom has increased
drastically since 2015. Number of cultivating maize had
increased until 2014 then decreased in 2015. Recently, numberof cultivating new cash crops such as cassava have increased.

Fig. 1: Change of number of households from 2001 to 2018 ineach livelihood activity in Bong village.
Fig. 2 shows that the villagers in Houay Su village mostly
cultivate upland rice and collect NTFPs. Paddy field had
expanded steadily along with the improvement of access and it
provided opportunity and flexibility to local people to expand
cash crops to get more income. Number of household
cultivating maize gradually increased until 2016, but
decreased drastically in 2017 and was followed by increase in
new cash crops such as coffee, cassava, cardamom andAmorphophallus sp. Almost all villagers cultivate coffee since2017 supported by Lao Z49 organization from the UnitedStates. They chose Houay Su village to cultivate coffee as analternative of opium poppy cultivation.

MATERIALS AND METHODS
The livelihood change is described based on statistic data
and by interviewing key informants of each household andperson in charge district related to local livelihood. After thecollection of both quantitative and qualitative data werecollecting through focus group discussion and individualinterviews. In addition to participatory observation relating toresearch objectives, seventy households of two villages werechosen and interviewed with semi-structured questionnaire. In
this study, wealth ranking methods were applied for analyzingcollected data and information.

86Proceedings of International Symposium on Animal Production and Conservation for Sustainable Development 2018
UGSAS-GU & BWEL Joint Poster Session on Agricultural and Basin Water Environmental Sciences 2018: P03

with traders and development projects. The people in Houay
Su village with good accessibility have many alternative cash
crops for sustainable livelihood. However, cultivating cash
crops related to expansion of agricultural land and it might beLQÀXHQFHV WKH HQYLURQPHQWDO FRQVHTXHQFHV RI H[SDQVLRQ DV
driver of deforestation; replace forests, degraded forests, or
grasslands (Gibbs et al. 2010).

ACKNOWLEDGMENTS
The authors would like to express sincere gratitude to all
farmers and officers of Sone district, Houaphan province. This
research could not be done without their cooperation.

REFERENCES
Gibbs, H. K., Ruesch, A. S., Achard, F., Clayton, M. K.,
Holmgren, K., Ramankutty, N., and Foley, J. A. (2010).
Tropical forest were the primary sources of new
agricultural land in the 1980s and 1990s. PNAS. vol.107
no. 38, Pages 16732-16737.
Rigg, J. (2006). Land, farming, Livelihoods, and Poverty:
Rethinking the Links in the Rural South. World
Development Vol. 34, No.1, Pages 180-202.
Sharkey, J. R., and Horel, S. (2008). Neighborhood
Socioeconomic Deprivation and Minority Composition
Are Associated with Better Potential Spatial Access to the
Ground-Truthed Food Environment in a Large Rural Area.
The Journal of Nutrition, Volume 138, Issue 3, 1 March
2008, Pages 620–627.
UNDP. (2004). Accessibility, rural roads and sustainable ruraldevelopment. Background paper for the road sector donor
coordination meeting, Vientiane, February 6-7.
Warr, P. (2006). The Impact of Road Development on Poverty
in the Lao People’s Democratic Republic. Asia-Pasific
Development Journal Vol.13 No.2.

Fig. 2: Change of number of households from 2001 to 2018 ineach livelihood activity in Houay Su village.

DISCUSSION
This study clarified that road construction has given impacts
directly to agricultural activities and livelihood changes. The
village committee in Bong village allocated 2 a per person perhousehold to all households, so, the household who have a lotof number of family, they will get more paddy area to provide
their food security. Even though the villagers had changes
their livelihood in the last decade, they still hold out tocultivate rice in upland field and collecting NTFPs especially
red mushroom as their livelihood strategies. On the other
hand, cultivating rice in upland field and collecting NTFPs are
the main livelihood in Houay Su village, and in the recent
years, agricultural land has expanded to include new cash
crops. The people in Bong village with bad accessibility did
not have alternative cash crops because of less opportunity

87Proceedings of International Symposium on Animal Production and Conservation for Sustainable Development 2018
UGSAS-GU & BWEL Joint Poster Session on Agricultural and Basin Water Environmental Sciences 2018: P04

Antibiotics resistance genes in vermicomposting of fruit and vegetablewaste added with excess activated sludge
Wenjiao Li 1, Guangyu Cui 1, Yasushi Ishiguro 2 and Fusheng Li 2
1. Graduate School of Engineering, Gifu University, Japan
2. River Basin Research Center, Gifu University, Japan
bed compartment. The image diagram of the reactors is shownas Fig.1. The reactors were divided into a control group and atest group. The bed compartment was filled with 100 g (wetbasis) of bed material, and then 10 earthworms (0.35 - 0.50
g/worm) were invited. For the treatment compartment of thecontrol reactors, 100 g (wet basis) of banana peels, cabbage,
lettuce, carrot, potato and sludge were added respectively. For
the treatment compartment of the test reactors, 100 g (wetbasis) of the mixture of each type of fruit and vegetable wastewith sludge were added respectively at the mixing ratio of 3:2(fruit and vegetable waste : sludge). All reactors were kept indark and were run under a constant temperature (25 Ԩ).

INTRODUCTION
Fruit and vegetable waste is generated during production,
processing and consumption. Conventional methods used fortreatment and/or recycling of fruit and vegetable waste include
incineration, landfill, anaerobic digestion, feeding andcomposting. Compared to these methods, vermicomposting
has the advantages of effective stabilization of the organic
waste through the combined involvement earthworms andbacteria in decomposition of the easily decaying organic
constituents and of higher reuse value for its final product as
fertilizers or soil conditioner.
On the other hand, activated sludge is the main byproduct ofsewage treatment process and is also an organic waste, which
is mainly consisted of microorganisms and can be treated byvermicomposting. The rich content of proteins in activatedsludge may promote the growth of earthworms, thus
improving the stabilization efficiency of fruit and vegetablewaste and may improve the value of the final products as
fertilizers due to the high content of nitrogen and phosphorus.
Our previous results on vermicomposting of fruit andvegetable waste indicated that adding excess activated sludge
could play a positive role in the growth of earthworms,
decomposition efficiency of fruit and vegetable waste and thefertilizer value of the final products.
However, numerous studies have identified that activatedsludge is a significant reservoir of antibiotics resistancebacteria (ARB) and antibiotics resistance genes (ARGs) due tooverused antibiotics and their metabolites discharged into thesewage system. Some researchers mentioned that ARGs mightproliferate in sludge due to the preferential survival andselection pressure of ARB during wastewater treatment.
Adding activated sludge into the vermicomposting systemtreating fruit and vegetable waste can bring ARGs into thesystem. As a result, depending on the fate and behavior of
ARGs in the vermicomposting process, the safety of the final
product as fertilizers may differ greatly. Nevertheless,information regarding the fate and behavior of ARGs duringvermicomposting of fruit and vegetable waste added withexcess activated sludge is not available.
Accordingly, the main objective of this study was toinvestigate the occurrence and behavior of ARGs duringvermicomposting of five types of fruit and vegetable waste
added with excess activated sludge.

10.1 cm

Plastic plate opened
with six holes(1 cm㽢 1 cm )14.8 cmTreatmentnt compartment

3.5 cm

Bed compartment
Leachate collection

Fig.1 Image diagram of each vermicomposting reactors.
2. DNA extraction and quantitative PCR (qPCR)
Frozen dried samples were used for extracting and purifying
total genomic DNA with the DNA extraction kit (MOBIO,
USA) based on manufacturer’s manual.
Typical ARGs targeted in this study included tet G, tet M,
tet X, sul 1 and qnr S. Class 1 integrase (intl 1) was also
targeted. The choice of these ARGs for study is because
tetracycline resistance genes, sulfonamides resistance genes
and quinolone resistance genes have been widely detectedfrom wastewater treatment plants due to the overuse of therelevant antibiotics for animal production and in human
medical care. The abundance of the ARGs, intl 1 and bacterial
16S rDNA gene was determined by the quantitative PCR
instrument (TP800, Takara, Japan). The standard DNA of tet
G, tet M, tet X, sul 1, qnr S and intl 1 were constructed by the
TA-cloning method (Takara, Japan). Each gene was quantified
in triplicate for each sample.

RESULTS AND DISCUSSION

MATERIALS AND METHODS

1. Abundance of ARGs and intl 1
Compared to the fruit and vegetable samples before adding
the sludge, the samples after mixing the sludge showedapparently higher values for the abundance of ARGs and intl 1
before the start of vermicomposting. As displayed in Fig. 2,
tet G in the treatment compartment increased from 3.82E+06,
5.27E+05, 4.39E+05, 1.00E+06, and 8.81E+05 copies/g-dry to
9.47E+07, 4.92E+07, 8.67E+07, 1.01E+08, and 8.98E+07
respectively in the treatment for banana peels, cabbage,
lettuce, carrot and potato after adding the sludge. The
observed increases can be easily explained as the result
brought about by rich tet G in the sludge (1.44E+08 copies/g-

1. Vermicomposting experiment
The earthworm species Eisenia foetida was used. Fruit andvegetable waste included banana peels, cabbage, lettuce,
carrot and potato. Excess activated sludge was collected froma slaughterhouse wastewater treatment plant and was used
after dewatered. A mixture of soil with final vermicompost
from our previous study was used as the bed material to avoid
the threat to earthworms’ survival from leachate generated
during vermicomposting. Eleven plastic containers were usedas the reactors for vermicomposting. Each reactor included
two compartments, namely the treatment compartment and the88Proceedings of International Symposium on Animal Production and Conservation for Sustainable Development 2018
UGSAS-GU & BWEL Joint Poster Session on Agricultural and Basin Water Environmental Sciences 2018: P04

dry). Similarly, significant increases were also observed forother genes except qnr S in the treatment for lettuce, as could
be seen from Table 1. For qnr S in the treatment for lettuce,
since the abundance of qnr S in lettuce (4.62E+05 copies/gdry) was closer to that in the sludge (5.27E+05 copies/g-dry),a significant increase after adding the sludge was not revealed.
After vermicomposting, for the treatment of fruit andvegetables added with the sludge, the results in Fig. 2 showedthat tet G in the treatment compartment decreased. For thetreatment without adding the sludge, tet G showed a generaltrend of increases for all fruit and vegetables. The values of
the abundance of tet G in the treatment compartment of thetreatment with the addition of the sludge were markedly
higher than the treatment without the addition of the sludge.
For tet M, sul 1, qnr S, and intl 1, similar results with tet G
were found in the treatment compartment after
vermicomposting. For tet X, however, the abundance in thetreatment compartment before vermicomposting fell in therange of 2.62E+04 – 2.38E+06 copies/g-dry for all reactors,
and increased to 6.94E+04 – 3.27E+07 copies/g-dry after
vermicomposting.
In this study, the material in the bed compartment after
vermicomposting was considered as the final product. No
significant changes of tet G were found after vermicompostingin all bed compartments except for the treatment for potato,
sludge, and the cabbage added with sludge, as could be seenfrom Fig.2. The changing extent of other ARGs and intl 1 in
the final product with the addition of sludge was shown in
Table 1. Apart from the changes revealed for intl 1, tet G, andsul 1 in the treatment for cabbage after adding sludge, and thechanges revealed for qnr S in the treatment for banana peels
after adding sludge, no significant changes were observed in
the final product of other treatments. Meanwhile, even if
abundant ARGs were introduced into the vermicomposting
system by adding sludge, the ARGs were not remained ortransferred into the final product during the vermicompostingprocess. Considering both the treatment compartment and bed
compartment, the reduction of ARGs was detected during thevermicomposting. Huang et al. (2018) reported a similar resulton the changes of tet C, tet G, tet M, tet O, tet W and tet X
during vermicomposting treatment of sludge, with the
reduction rate of the genes being 83.1%, 39.6%, 99%, 80.2%,
94.1% and 60.9%, respectively. The reduction is probably due
to the reason that earthworms regulated the bacterial
community and their environment in vermicomposting system.
2. Abundance of 16S rDNA gene
The abundance of 16S rDNA reduced significantly in alltreatment compartments after vermicomposting except thetreatment for banana peels, cabbage and its mixture withsludge. The largest decrease occurred in the treatment for
carrot (from 3.27E+10 copies/g-dry to 8.52E+09 copies/gdry), followed by the treatment for sludge (from 2.44E+10
copies/g-dry to 5.70E+09 copies/g-dry). A previous study
reported decreases in the abundance of 16S rDNA for all
treatments of excess activated sludge with three different
earthworm densities. The decreases are probably due to therapid depletion of nutrients by the intensified digestion ofearthworms (Cui et al. 2018). In contrast, different results
were observed in the bed compartment of the treatments forbanana peels, potato, the mixture of banana peels and sludge,
the mixture of lettuce and sludge. The most significant
increase occurred in the bed compartment of the treatment for
potato (from 2.76E+10 copies/g-dry to 3.72E+10 copies/gdry). Huang et al. (2018) revealed an increase of 16S rDNA
during vermicomposting due to the effect of earthworms inenriching the agricultural probiotics in the final
vermicomposting product. It is worth to notice that asignificant decrease (from 2.76E+10 copies/g-dry to 1.75E+10
copies/g-dry) in the bed compartment was detected for thetreatment for cabbage. The significant difference of the ARGs
in the bed compartment for the treatment of cabbage with andwithout the addition of sludge shown in Table 1 can probably
be explained by the changes of 16S rDNA.

tet G in treatment compartment(copies/g-dry)

1.60E+08

InitialFinal

InitialFinal

1.40E+08
1.20E+08
1.00E+08
8.00E+07
6.00E+07
4.00E+07
2.00E+07
0.00E+00

1.40E+08

tet G in bed compartment(copies/g-dry)

1.20E+08
1.00E+08
8.00E+07
6.00E+07
4.00E+07
2.00E+07
0.00E+00

Fig. 2 Changes of tet Gin the treatment compartment andbed compartment before and after vermicomposting. Data
are represented as mean and standard deviation (n = 3).
Table 1The changing extent of ARGs and intl 1 after
adding sludge in the treatment compartment before
vermicomposting and the bed compartment after
vermicomposting. Values show the percentage of changes
in the treatment compartment, and the values in theparentheses show the percentage of changes in the bed
compartment.

Bananapeels
Cabbage
Lettuce
CarrotPotatointl 1
(%)

tet G
(%)

tet M
(%)

tet X
(%)

sul 1
(%)

qnr S
(%)

816.9
(19.6)
5028.7(104.9)11543(-45.0)181.5(-20.4)66016(-17.0)2376.9(-3.9)
9247.3(143.9)19647(-5.4)
9964.3(-12.6)10095(-26.9)2398.4(0.9)
3089.1(44.0)
33684(35.0)
6077.3(-35.1)
20682(19.3)

1779.6(22.5)
2915.1(63.5)
4382.8(-68.7)5327.1(-56.3)4745.3(-28.4)2108.0(51.9)
34136(335.4)71718(-41.9)4691.8(-25.5)74554(-26.0)1195.2(684.8)100.7(-55.7)-1.2(186.6)671.3(-7.3)
4014.3(22.4)REFERENCES
Kui Hang et al. (2018) Effects of earthworms on the fate of
tetracycline and fluoroquinolone resistance genes of sewage
sludge during vermicomposting. Bioresource Technology
259: 32-39.
Guangyu Cui et al. (2018) Changes of quinolone resistancegenes and their relations with microbial profiles duringvermicomposting of municipal excess sludge. Science ofTotal Environment 644: 494-502.

89

Proceedings of International Symposium on Animal Production and Conservation for Sustainable Development 2018
UGSAS-GU & BWEL Joint Poster Session on Agricultural and Basin Water Environmental Sciences 2018: P05

Mating-type diversity in Phytophthora colocasiae causing leaf-blight oftaro in Japan
Wenzhuo Feng1, Kayoko Otsubo2, Ayaka Hieno2, Haruhisa Suga3 and Koji Kageyama2
1
The United Graduate School of Agricultural Science, Gifu University, 1-1 Yanagido, Gifu 501-1193, Japan
2
River Basin Research Center, Gifu University, 1-1 Yanagido, Gifu 501-1193, Japan
3
Life Science Research Center, Gifu University, 1-1 Yanagido, Gifu 501-1193, Japan

incubating alone were examined twice after more than amonth to determine whether the isolate is heterothallic or
homothallic based on the formation of oospores.

INTRODUCTION
Taro (Colocasia esculenta (L.) Schott) is one of the mostpopular staple crops for its edible corms and leaves in Japan.
Recently in Japan, especially in Ehime, Miyazaki andKagoshima prefectures where are the major production areas
for taro, the disease of taro leaf blight has occurred since 2014
and is expanding year by year. The causal agent was isolated
from diseased leaves and identified as Phytophthora
colocasiae Raciborski. The pathogen can asexually produce
zoosporangia, and zoospores released from them can spread
rapidly via water during the frequent heavy rain and typhoon
period. It has been reported that in naturally infested soils, thesoil survival of P. colocasiae sporangia is less than 21 days,
lasts for 3 months in taro leaves, and lasts for more than 107
days in soil at –1,500 J/kg matrix potentials. Therefore, in
addition to zoosporangia, the mating-type of P. colocasiae
isolates that is related to long-lived oospore formation is very
important for determining a primary inoculum.
Phytophthora colocasiae has been reported in many
locations, such as Southeast Asia and the Pacific. The species
was previously described as only existing heterothallic
isolates, depending on the presence of opposite mating types(A1 and A2) to form oospores, until Lin and Ko (2008) foundseven homothallic A1A2-type isolates in Taiwan. However,
oospores seemed difficult to form in most of the areas becauseof non-coexistence between A1 and A2 type there (Narula and
Mehrotra 1980; Ann et al 1986; Tyson and Fullerton 2007;
Mellow et al. 2018). Up to now, there is no report about themating type of P. colocasiae in Japan. Therefore, in this study,
our objectives were to investigate the mating-type diversity,and analysis their distribution in taro fields in Japan.

RESULTS AND DISCUSSION
Mating-type diversity
In all, there were five kinds of mating types confirmed fromthe 150 isolates: two kinds of heterothallic isolates of A1 andA2 types, and three kinds of homothallic isolates thatgenerated oospores in the plates of incubation alone,
containing A1 type which generated oospores with pairing of
A2 strain, A2 type which generated oospores with A1 strain,
and A1A2 type which generated oospores with both of A1 andA2 strains. This complex mating-type diversity in P.
colocasiae has not been reported all over the world.
Mating-type distribution in taro fields
Table 1. The mating-type distribution in three prefectures
Location

Kagoshima

Miyazaki

Ehime

1
2
3
4
5
Total
1
2
3
4
5
6
7
8
9
Total
1
2
Total

Homothallic a

Heterothallic

Number offieldsNumber ofisoltates

A1

A2

A1

A2

A1A2

1
2
2
1
3
9
2
1
6
14
1
2
1
1
2
30
14
1
15

5
3
14
6
31
59
3
1
9
24
1
3
1
1
6
49
41
1
42

0
0
0
1
0
1
0
0
0
1
0
0
0
0
0
1
6
0
6

2
2
5
2
11
22
3
1
5
12
1
1
1
1
3
28
24
1
25

0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0

2
0
9
2
20
33
0
0
4
10
0
2
0
0
3
19
9
0
9

1
1
0
1
0
3
0
0
0
1
0
0
0
0
0
1
2
0
2

a
Homothallic isolates that generated oospores in the plates of incubation alone, containing A1 type which generated oosporeswith pairing of standard A1 type, A2 type which generated oospores with standard A2 type, and A1A2 type which generated
oospores with both of standard A1 and A2 types.

MATERIALS AND METHODS

Totally, we investigated the mating-type of 150 isolates
from 54 fields in Japan (Table 1). One, 22 and 36 out of 59
isolates in Kagoshima, 1, 28 and 20 isolates out of 49 isolates
in Miyazaki, and 6, 25 and 11 out of 42 isolates in Ehime
prefecture were identified as A1 type, A2 type andhomothallic, respectively (Table 1). These results showedthat in Japan the most isolates were A2 type, only a few
isolates were A1 type and more than 44% isolates were
homothallic. On the other hand, 23 of 32 fields, where two or
more isolates were collected, were found multiply infested

Collection and maintenance of isolates
A total of 150 isolates were isolated and obtained from thediseased taro leaves and petioles of 9 fields in Kagoshima, 30
fields in Miyazaki and 15 fields in Ehime prefecture (Table 1).
All isolates were maintained on corn meal agar or potatodextrose agar media at 20°C in the dark.
Mating type testingAll isolates were inoculated on 5 ml V8 medium (1 ml V8
juice, 12.5 mg CaCO3, 0.1 g agar and 4 ml distilled water) at
25°C in darkness, respectively. Two heterothallic isolates
obtained from taro fields (EPC201522 and EPC2017Ko1)
were selected as standard type strains and strictly verified thatthe former isolate is A1 mating type and the latter is A2
mating type.
Totally, three V8-medium plates were prepared for eachisolates, one for incubating alone, and the other two for
pairing it with A1 strain of EPC201522sh1 and A2 strain of
EPC2017K01sh, respectively. The oospores in plates forpairing were examined after incubation for 6 days and 10
days. The absence of oospores at the interface between
colonies indicated the same mating type, while the presence of
oospores indicated the opposite mating type. The plates for

Table 2. The mating types in fields where two or
more isolates were collected from
Mating types
A1 only
A2 only
Homothallic only
A1and A2A1 and homothallic
A2 and homothallic
A1, A2 and homothallic
Total

90

Number of fields
0
8
1
2
0
16
5
32

Proceedings of International Symposium on Animal Production and Conservation for Sustainable Development 2018
UGSAS-GU & BWEL Joint Poster Session on Agricultural and Basin Water Environmental Sciences 2018: P05

with A1 and A2, A2 and homothallic, or A1, A2 andhomothallic (Table 2). Hence, this complex and similarmating-type distribution in the three prefectures indicated thatthe possibility of the cross-infestation of P. colocasiae inJapan would be very high.

In this study, a complex mating-type diversity in P.
colocasiae, including heterothallic isolates of A1 and A2 types
and homothallic isolates of A1, A2 and A1A2 types waspresented in Japan. This situation might be one of the reasonsthat the disease are expanding. The oospores with longevity
and durability generated easily can survive in severe
environment safely such as winter. The sexual reproduction
would introduce a longer-lived form of isolate and increase thechances of generating new and potentially more threatening P.
colocasiae genotypes.

Mating types in one leaf
Two or three isolates were collected from the different
disease spots in each of 13 leaves. Based on the analysis ofmating-type, as shown in Table 3, a total of eight leaves were
infected with multiple mating types, including one leaf
infected with A1 and homothallic and seven infected with A2
and homothallic. This results indicated the disease spots in one
leaf could be caused by different source of P. colocasiae.

ACKNOWLEDGMENTS
This study was supported by “Grant-in-Aid from the
Ministry of Agriculture, Forestry, and Fisheries of Japan”. We
thank T. Matsuda, H. Shibata, Z. Wang, K. Nakagawa, Y.
Murakami, Y. Mouri and T. Yamamoto in Ehime Prefectural
Government Agriculture, Forestry, S. Kuroki, Y. kushima, K.
Kuniki and K. Shimodo in Miyazaki Prefectural Agricultural
Experiment Station, Y. Nishi, K. Nishioka and T. Yuda in
Kagoshima Prefectural Institute for Agricultural Development,
and T. Tsukihoshi in The Western Region Agricultural
Research Center, National Agricultural and Food Research
Organization for providing isolates of P. colocasiae.

Table 3. The mating types of P. colocasiae in each leaves
Location
1

2

Kagoshima

34

5

Heterothallic

Homothallic

No.(leaf)

Number ofisolates

A1

A2

A1

A2

A1A2

1

3

0

2

0

1

0

2

2

1

0

0

1

0

1

3

0

2

0

1

0

2

3

0

3

0

0

0

3

3

0

1

0

2

0

1

3

0

1

0

2

0

2

3

0

0

0

3

0

3

2

0

1

0

2

0

1

2

0

1

0

1

0

2

3

0

0

0

3

0

1

3

0

0

0

3

0

2

2

0

0

0

2

0

3

3

0

1

0

2

0

REFERENCES
Ann, P. J., Kao, C. W., & Ko, W. H. (1986). Mating-type
distribution of Phytophthora colocasiae in Taiwan.
Mycopathologia, 93(3), 193-194.
Lin, M. J., & Ko, W. H. (2008). Occurrence of isolates of
Phytophthora colocasiae in Taiwan with homothallic
behavior and its significance. Mycologia, 100(5), 727-734.
Mellow, K. D., Tyson, J. L., Fullerton, R. A., Tugaga, A., &Maslen-Miller, A. (2018). Mating types of Phytophthora
colocasiae on the island of Upolu, Samoa. New Zealand
Plant Protection, 71, 289-292.
Narula, K. L., & Mehrotra, R. S. (1980). Occurrence of A1
mating type of Phytophthora colocasiae. Indian
Phytopathology, 33(4), 603-604.
Tyson, J. L., & Fullerton, R. A. (2007). Mating types of
Phytophthora colocasiae from the Pacific region, India andSouth-east Asia. Australasian Plant Disease Notes, 2(1),111-112.

Segregation of mating type in one isolate
We selected two heterothallic isolates of each A1 and A2
types and six homothallic isolates for single-hypha separation(Table 4). Six single-hyphae cultures segregated from the two
A1 isolates and 15 single-hyphae cultures from the two A2
isolates were confirmed as the same type with their own
original isolates. Among the 19 single-hyphae cultures fromfive homothallic A2 type isolates, four kinds of types,
including heterothallic A1, A2 and homothallic A2, A1A2
types were found. The other one homothallic A1A2 isolate
segregated into 12 single-hyphae cultures. Three, one, sevenout of the 12 cultures were confirmed as heterothallic A1, A2
and homothallic A2 types, respectively. In addition, theremaining one culture was confirmed as homothallic A1 typethat was firstly found (Table 4). These results suggest that thehomothallic isolates will include both of heterothallic andhomothallic features, and that heterothallic isolates will have astable property.
Table 4. The mating-type analysis in single-hyphae cultures ofdifferent type isolates
Type

Number of
single-hyphaecultures

A1

A2

A1

A2

A1A2

EPC201509

A1

2

2

0

0

0

0

EPC201522

A1

4

4

0

0

0

0

KS17Ai1-2

A2

10

0

10

0

0

0

EPC2017Ko1

A2

5

0

5

0

0

0

EPC201527

A2

4

0

1

0

2

1

EPC201534

A2

6

0

0

0

6

0

KS16TaOKI3

A2

3

1

2

0

0

0

KS16TaOKI4

A2

3

0

0

0

2

1

KS16TaOki5

A2

3

0

1

0

2

0

KS16TaYo2

A1A2

12

3

1

1

7

0

IsolateHeterothallic

Homothallic

Heterothallic

Homothallic

91

Proceedings of International Symposium on Animal Production and Conservation for Sustainable Development 2018
UGSAS-GU & BWEL Joint Poster Session on Agricultural and Basin Water Environmental Sciences 2018: P06

Estimation on nitrogen deposition into Takayama forest over three years
Ruoming Cao1, Siyu Chen1, Shinpei Yoshitake2 and Toshiyuki Ohtsuka2
1.
The United Graduate School of Agricultural Science, Gifu University
2.
River Basin Research Center, Gifu University
Calculation– N fluxes were calculated based on volume andconcentrations. In order to evaluate the interactions of canopy
INTRODUCTION
to N deposition, we used net TF. Net TF was performed interms of flux data: Net TFa = TFa + SF – BP, Net TFb = TFb –Nitrogen (N) will input into forest ecosystems from
TFa. In this study, stemflow of dwarf bamboo was neglected.
atmosphere by precipitation which is called N deposition informs of dissolved organic nitrogen (DON) and dissolved
inorganic nitrogen (DIN). Nowadays, more and more N input
RESULTS
into ecosystems mostly due to human activities, such as
agricultural and industrial activities. There are various
N deposition via bulk precipitation–About 11.5 kg N ha–1
influences of N deposition on forests. Increasing N depositionyr–1 of dissolved N was deposited into the study site by BP
could have a rise in biomass production by 0.31 Pg C yr–1
with 32 % snowfall flux. The percentage of DON to TDN(Thomas et al. 2010) and canopy uptake or leaching occurred
amounts to 72 % (Table 1).
in response to N deposition (Schwarz et al. 2014; IzquietaRojano et al. 2016). On the other hand, the environmental
Temporal dynamics of N deposition–Average monthly
consequences are also serious faced with increased N
concentrations of DON in BP followed a clear seasonal patterndeposition, for example, nitrate leaching to groundwaterin 2015 and 2016, with the highest concentration on May 2015
(Gundersen et al. 2006). Studies on N deposition in Europe
or June 2016 and tended to decrease over subsequent time.
and China have evolved well, especially focusing on dissolved
However, there was no clear seasonal pattern in 2017 (Fig.1).
inorganic nitrogen (DIN) deposition which is known to have
The dynamics of DIN concentrations revealed decreasing
anthropogenic source. And dissolved organic nitrogen (DON)
trends from spring to winter in 2015 and 2017. But there was
gradually is attracting attention as significant part ofno clear seasonal pattern in 2016 (Fig.1). For average DON
atmospheric N deposition (Cornell 2011). There are few
flux, higher flux was deposited during summer, except 2017
studies on N deposition in Japanese forests.
when N deposition in autumn also higher (Fig. 1).
In Takayama forest site which is an Asia flux network
Canopy exchange–There was positive relationship between
forest, compartment models of carbon pools and fluxes haverainfall and DON fluxes in net TFa but negative relationshipindicated (Ohtsuka et al. 2007; Chen et al. 2017). It has
become clear that where and how the forest stores carbon.
between water fluxes in TFb and DON Fluxes in net TFb (P <But there is no monitoring the N fluxes by N deposition. It is0.05) (Table 2). It suggested that DON was leached by tree
not yet clear that temporal dynamics and effect of N
canopy however uptake by bamboo canopy. For DIN flux,
deposition in the forest site. This study aims to estimate DON
there was significantly positive correlation between rainfall
and DIN deposition input into Takayama forest during threeand DIN fluxes in TFa (P < 0.05), but no significant
years and to identify level and dynamics of N deposition
correlation existed between water fluxes in TFb and DIN
during growing season and snow season, and response offluxes in net TFb (Table 2). It turned out that DIN flux was
canopy to N input into forest.
leached by tree canopy, whereas dry deposition significantly
influenced DIN flux in TFb.

MATERIALS AND METHODSDISCUSSION

Study site–The study site is a cool-temperate deciduous
broad-leaved forest (Takayama forest) in central Japan(36°08’N, 137°25’E, 1420 m a.s.l.). Dwarf bamboo densely
cover the forest floor. The climate is cool-temperate withgrowing season (May–Nov) and snow season (Dec–April).

N deposition–DIN flux was within the range of Japaneseand European studies (Cape et al.2012; Mitchell et al. 1997),
whereas lower than that in China (Jia et al. 2014), where is
faced with serious environmental problems. DON flux
coincide with that in Guangzhou city in China but at higher
level compared to Europe (Li et al. 2012; Cape et al. 2012).
DON contribution in Takayama forest was greater than moststudies (2 % ~ 28 % in European forests, Cape et al. 2012;
7 % ~ 67 % in Chinses forests, Zhang et al. 2012), but was inaccordance with that in the remote Tibetan area of China(Zhang et al. 2008). A major contribution of DON to TDN in
the study site showed that importance of DON deposition in
the forest site. Seasonal pattern of DON concentration maybe
due to nitrogen fertilizer use in spring, which was also
reported by Izquieta-Rojano et al. (2016) who argued that
DON deposition was mainly dominated by agriculturalactivities. And snow contribution cannot be ignored based on
the characteristic of snow season existed.

Experimental setup—During growing season (May–Nov),
we set up the samplers of bulk precipitation (3 replicates) near
1 ha plot, samplers of throughfall above bamboo (TFa) (9replicates) and throughfall below bamboo (TFb) (9 replicates),and samplers of stemflow (SF) (9 replicates) within 1 ha plot.
The volumes of water samples were measured by cylinder(5L) or rain gauge and water subsamples were collected by
100 ml bottles at once per month from 2016 to 2018. Duringsnow season, we collected snow samples (3 points) at each 10
cm distance from snow surface to soil surface and transformed
melted sample to 100ml bottles.
Chemical analysis—$IWHU EHLQJ ILOWHULQJ E\ ȝP
membrane filter, we measured the concentrations of total
dissolved nitrogen (TDN), NH4+ and NO3–+NO2– (DIN) by a
QuAAtro 2-HR nutrient analyzer. DON was calculated bydifference between TDN and DIN.

Canopy exchange–The leaching of DON by tree canopy is
general. However, DON uptake is less reported (IzquietaRojano et al. 2016). DON was uptake by understory bamboo
canopy implied that DON flux was an important source for92

Proceedings of International Symposium on Animal Production and Conservation for Sustainable Development 2018
UGSAS-GU & BWEL Joint Poster Session on Agricultural and Basin Water Environmental Sciences 2018: P06

nutrient supply in the study site.

Jia, Y.L., Yu, G.R., He, N.P., Zhan, X.Y., Fang, H.J., Sheng,
W.P., Zuo, Y., Zhang, D.Y. and Wang, Q.F. (2014) Spatial
and decadal variations in inorganic nitrogen wet depositionin China induced by human activity. Scientific Reports
4:3763.
Li, J., Fang, Y.T., Yoh, M., Wang, X.M., Wu, Z.Y., Kuang,
Y.W. and Wen, D.Z. (2012) Organic nitrogen deposition inprecipitation in metropolitan Guangzhou city of southern
China. Atmospheric Research 113:57–67.
Mitchell, M.J., Iwatsubo, G., Ohrui, K. and Nakagawa, Y.
(1997) Nitrogen saturation in Japanese forests:
anevaluation. Forest Ecology and Management 97:39–51.
Ohtsuka, T., Mo, W., Satomura, T., Inatomi, M., Koizumi, H.
(2007) Biometric based carbon flux measurements and net
ecosystem production in a temperate deciduous broadleaved forest beneath a flux tower. Ecosystems 10, 324–334.
Schwarz, M.T., Bischoff, S., Blaser, S., Boch, S., Schmitt, B.,
Thieme, L., Fischer, M., Michalzik, B., Schulze, E.,
Siemens, J. and Wilcke, W. (2014) More efficient
aboveground nitrogen use in more diverse central European
forest canopies. Forest Ecology and Management. 313:274–
282.
Tomas, R.Q., Canham, C.D., Weathers, K.C. and Goodale,
C.L. (2009). Increased tree carbon storage in response tonitrogen deposition in the US. Nature Geoscience 3.
Zhang, Y., Song, L., Liu, X.J., Li, W.Q., Lu, S.H., Zheng,
L.X., Bai, Z.C., Cai, G.Y., Zhang, F.S. (2012) Atmosphericorganic nitrogen deposition in China. Atmospheric
Environment 46:195–204.
Zhang, Y., Zheng, L.X., Liu, X.J., Tim, J., Cape, J.N.,
Goulding, K., Fangmeier, A., Zhang, F. (2008) Evidence fororganic N deposition and its anthropogenic sources in China.
Atmospheric Environment 42:1035–1041.

ACKNOWLEDGMENTS
We are very grateful to the members of the Takayama
Forest Research Station and Ohtsuka lab for field work
support.

REFERENCES
Chen, S., Yoshitake, S., Iimura, Y., Asai, C., Ohtsuka, T.
(2017) Dissolved organic carbon (DOC) input to the soil:
DOC fluxes and their partitions during the growing season
in a cool-temperate broad-leaved deciduous forest, central
Japan. Ecol. Res. 32, 713–724.
Cornell, S.E. (2011) Atmospheric nitrogen deposition:
Revisiting the question of the importance of the organic
component. Environmental Pollution 159:2214–2222.
Cape, J.N., Tang, Y.S., Gonzalez-Bentez, J.M., Mitosinkova,
M., Makkonen, U., Jocher, M. and Stolk, A. (2012) Organic
nitrogen in precipitation across Europe. Biogeosciences9:4401–4409.
Gundersen, P., Schmidt, I.K., Raulund-Rasmussen, K. (2006)
Leaching of nitrate from temperate forests – effects of air
pollution and forest management. Environmental Reviews
14:1–57.
Izquieta-Rojano, S, García-Gomez, H, Aguillaume, L,
Santamaría, J.M., Tang, Y.S., Santamaría, C., Valiño, F.,
Lasheras, E., Alonso, R., Àvila, A., Cape, J.N., Elustondo,
D. (2016) Throughfall and bulk precipitation of dissolved
organic nitrogen to holm oak forests in the Iberian
Peninsula: Flux estimation and identification of potential
sources. Environmental Pollution 210:104–112.

Table 1. Average nitrogen concentration and average annual flux in bulk
precipitation during three years (May 2015–April 2018). Values inparentheses are standard deviation (n = 3). Different letters are
significantly different between concentrations or fluxes of dissolved
nitrogen in snowfall and rainfall (P < 0.05). DIN = Dissolved inorganic
nitrogen, DON = Dissolved organic nitrogen, TDN = Total dissolved
nitrogen.
RainfallSnowfallTotal(kg N ha–1 yr–1)

Snowfall/Total0.39 (0.03) a
0.13 (0.04) a
0.53 (0.04) a
1543 (17.7)

0.32 (0.16) a
0.23 (0.09) a
0.53 (0.19) a
670 (125)

–
–
–
–

–
–
–
–

5.96 (0.52) a
1.66 (0.04) a
7.61 (0.55) a
0.78 (0.01)

2.28 (1.45) b
1.56 (0.79) a
3.84 (1.96) a
0.58 (0.11)

8.24 (1.01)
3.22 (0.76)
11.5 (1.42)
0.72 (0.05)

0.27 (0.14
0.46 (0.15
0.32 (0.13––1Concentration (mg L )
DON
DIN
TDN
Precipitation (mm)Flux
(kg N ha–1 period–1)
DON
DIN
TDN
DON/TDN

Table 2. Spearman correlation between rainfall amount (mm) and net
throughfall above bamboo (Net TFa) (kg N ha–1 episode–1) or between water
depth of TFa (mm) and net throughfall below bamboo (Net TFb) (kg N ha–1
episode–1) (n = 23). DIN = Dissolved inorganic nitrogen, DON = Dissolved
organic nitrogen. * means significant at P < 0.05.

Net TFa
Net TFb

NH4–N
0.14
–0.03

NO3–N
0.12
0.08

DIN
0.42*
0.20

DON
0.52*
–0.46*

93

(a)

(b)

Fig. 1. Mean monthly concentration (mg L–1)(a) and mean flux (kg N ha–1 episode–1) (b) inbulk precipitation from May 2015 to April
2018. Shaded areas show snow season. Fluxes
on January 2016, January 2017 and March2018 stand for fluxes during snow period
between two adjacent years. Error bars show
the standard error (n=3). DON = Dissolved
organic nitrogen, DIN = Dissolved inorganic
nitrogen.

Proceedings of International Symposium on Animal Production and Conservation for Sustainable Development 2018
UGSAS-GU & BWEL Joint Poster Session on Agricultural and Basin Water Environmental Sciences 2018: P07

Immobilization of cesium in contaminated forest soil using various
additives: evaluation based on the inhibition effect on its transfer to grass
Huijuan Shao1 and Yongfen Wei1, 2
1. The United Graduate School of Agricultural Science, Gifu University, Japan
2. River Basin Research Center, Gifu University, Japanof mesh. The basic physicochemical properties of soil andadditives are summarized in Table 1. As shown, soil had the
INTRODUCTION
lowest values of pH, cation exchange capacity (CEC) andspecific surface area (SSA); while biochar had the highest
Soil contamination of radionuclides released from nuclear
values of pH, organic matter content, CEC and SSA.
accidents and nuclear tests has been a serious environmental
problem in the world (Ashraf et al. 2014). Due to theExperimental design—Stable 133Cs was used in this study
Fukushima Daiichi Nuclear Power Plant accident on Marchsince
its chemical behavior for vegetation was reported to be
11, 2011, a large amount of radionuclides was released into
similar with that of 137Cs (Burger et al., 2018). CsCl solution
the northeastern part of Japan. Cesium-137 (137Cs) is of a
was uniformly mixed with soil and the Cs contamination levelspecial concern due to its long half-life (about 30 years), highwas set to be 100 mg/kg dry weight. 480 g of soil was placedsolubility in water and strong affinity to soil. Due to its
in each plastic pot and the soil was then well mixed with BC,
biogeochemical similarities with potassium (Zhu and
ISSA or Zeolite at a ratio 1:10 (additive: soil) by hand until
Smolders, 2000), many studies have reported that vegetation
homogeneity was achieved. In addition, no additive applied
takes in deposited 137Cs from soil through their roots, andsoil was used as control for comparison. Each treatment had
translocate it quickly to the leaves.
three replicates.
A majority of the land heavily contaminated with 137Cs innortheastern areas of Japan is covered by forests (HashimotoPot experiment and analysis—Napier grass (Pennisetum
et al. 2012). Extensive decontamination measures including
purpureum) was selected and cultivated inside a glass
topsoil removal, landfilling and soil washing have been
greenhouse in Gifu University. During the whole cultivation
conducted in Fukushima prefecture. These measures are
period from 2017/11/02 to 2018/06/02, the maximum andhighly effective at reducing the degree of contamination, but
minimum temperatures in the greenhouse were 40 °C and –undoubtedly they will cause environmental destruction and are
3 °C, respectively, and no additional fertilizer was applied.
too expensive to be implemented in the large scale of
After harvest, the grasses were washed carefully, separated
contaminated forest. Therefore, to develop an effective, ecointo three parts (leaf blade, leaf sheath and root), then freezefriendly and low-cost technology for treatment of 137Csdried, shattered by a crusher and digested with nitric acid in an
contaminated forest soil is strongly desired.
autoclave. The Cs concentration of the digested samples was
In-situ immobilization, which aims to inhibit the transfer of137
measured using inductively coupled plasma massCs from soil to vegetation and water bodies by increasing
spectrometry (ICP-MS, Agilent 7700X). The distribution ofcompetitive ion concentration or fixation capability by
Cs in the dried grass sample was analyzed using scanningapplication of certain additives is a promising technology (Leeelectron microscopy with energy dispersive X-ray
et al. 2009). To generate information that can be used forspectroscopy (SEM/EDX, Hitachi SU3500). EDX analysis
immobilization technology, the evaluation of inhibition effectwere performed at the acceleration voltage of 20 kV.
of three low-cost adsorbent additives on Cs transfer from soil
to vegetation was performed in this study. Meanwhile, thedistribution of Cs on different parts of vegetation was also
RESULTS AND DISCUSSION
examined.
Cesium concentration accumulated in different parts of the
MATERIALS AND METHODS
grass—As shown in Fig. 1, as compared with control (without
additive), all three additives obviously declined Cs
Soil and additive samples—Soil was collected from theconcentration in the grass. And especially biochar (BC)surface layer (0–15 cm depth) after removing plant and litter
showed the lowest value; the Cs concentration in the grass waslayers in the evergreen coniferous forest located along Ijira
reduced by 93.5 - 97.1%. The increase of potassium
Lake in Gifu Prefecture, Japan. Three additives including
concentration after additive addition is considered to be thecoconut shell biochar (BC), incinerated sewage sludge ash
most likely reason for this reduction due to the increased(ISSA, before phosphorus recovery), and natural zeolite
fixation capability of potting soil after additive addition(Zeolite) were obtained from TAKII Seed Corporation, North
increased (data not shown). Among different parts of Napier
Wastewater Treatment Plant of Gifu City, and SINKOU
grass, leaf blade possessed the highest Cs concentration. ThisSUNRISE Corporation, respectively. Soil and additives weremay indicate that for Napier grass the leaf blade was the main
all air-dried at room temperature and then sieved with a 2 mmorgan to accumulate Cs (Kang et al. 2012).
Table 1 Physicochemical properties of the soil and additives used in this study
Property

Soil

Biochar

pH (H2O)

4.88 ± 0.13

9.25 ± 0.21

5.27 ± 0.15

6.23 ± 0.11

Organic matter (%)
Cation exchange capacity(cmol/kg)
Specific surface area (m2/g)

15.85 ± 0.87

81.31 ± 2.14

0.27 ± 0.07

0.38 ± 0.07

24.77 ± 2.35

47.83 ± 2.36

30.15 ± 1.18

87.58 ± 2.35

21.63 ± 1.25

157.14 ± 3.29

34.93 ± 1.86

43.39 ± 2.51

94

Incinerated sewage sludge ash

ZeoliteProceedings of International Symposium on Animal Production and Conservation for Sustainable Development 2018
UGSAS-GU & BWEL Joint Poster Session on Agricultural and Basin Water Environmental Sciences 2018: P07

Cs concentration (mg/kg)

1400
1200

leaf blade

leaf sheath

BC

ISSA

root

1000
800
600
400
200
0
Control

ZeoliteFig. 1 Cesium concentration in different parts of grass
under different additive additions (BC: biochar, ISSA:
incinerated sewage sludge ash)Distribution of cesium in different parts of the grass—
According to Fig. 2 and Fig. 3 obtained from SEM/EDX
analysis, it is obvious that Cs was unevenly distributed indifferent parts of the grass, and its concentration inside theroot was higher (0.15%) than that on the outside surface(0.08%). Xylem (plant vascular tissue) inside the root is
considered to have mainly contributed to the result since it
conveys water and dissolved minerals from the roots to therest of the grass.
Leaf blade

Cs: 0.15%

300 PmFig. 3 Cesium distribution inside the grass root (crosssection; Cs: 100 mg/kg; control: without additive)
In addition, in biochar addition case, the Cs concentrations
on all parts of the grass (0.02% in average) were lower but thepotassium (K) concentrations were higher by comparing withthat of control (data not shown). It indicated that the increased
K concentration via biochar addition was one likely reason to
inhibit Cs uptake by grass root due to the competition between
Cs and K.

㻌REFERENCES

50 Pm

Ashraf, M., Akib. S., Maah, M., Yusoff, I., and Balkhair, K.
(2014) Cesium-137: radio-chemistry, fate, and transport,
remediation, and future concerns, Environmental Science
Burger, A., and Lichtscheidl, I. (2018) Stable andradioactive cesium: A review about distribution in theenvironment, uptake and translocation in plants, plant
reactions and plants' potential for bioremediation. Scienceof the Total Environment, 618, 1459–1485.
Hashimoto, S., Ugawa, S., Nanko, K., and Shichi, K. (2012)
The total amounts of radioactivity contaminated materials inforests in Fukushima, Japan. Scientific Reports, 2, 416,
10.1038/srep00416
Kang, D.J., Seo, Y.J., Saito, T., Suzuki, H., and Ishii, Y.
(2012) Uptake and translocation of cesium-133 in Napiergrass (Pennisetum purpureum Schum.) under hydroponic
conditions. Ecotoxicology and Environmental Safety, 82,
122–126.
Lee, S.H., Lee, J.S., Choi, Y.J., and Kim, J.G. (2009) In situ
stabilization of cadmium-, lead-, and zinc-contaminated soil
using various amendments. Chemosphere, 77, 1069–1075.
Zhu, Y.G., and Smolders, E. (2000) Plant uptake ofradiocaesium: a review of mechanisms, regulation andapplication. Journal of Environmental Botany, 51, 1635–
1645.

Cs: 0.09%㻌
0 09%

Leaf sheath 㻌

Cs: 0.08%㻌
100 PmRoot

Cs: 0.08%㻌
100 PmFig. 2 Cesium distribution on the outside surface ofdifferent parts of the grass (Cs: 100 mg/kg; control)95

Proceedings of International Symposium on Animal Production and Conservation for Sustainable Development 2018
UGSAS-GU & BWEL Joint Poster Session on Agricultural and Basin Water Environmental Sciences 2018: P08

Hydrological characteristics under different forest types in central Japan
Ali Rahmat1, Keigo Noda2, Kengo Ito2 and Masateru Senge2
1. The United Graduate School of Agricultural Science, Gifu University, Japan
2. Faculty of Applied Biological Sciences, Gifu University, JapanIn Fig. 2 showed, the peak discharge, direct runoff, runoff
coefficient in No.12 deciduous broadleaf forest is lower thanin No.10 evergreen coniferous forest, but relationship between
precipitation and direct runoff in the No.10 evergreen
coniferous is closer compared in No.12 deciduous broadleaf.
In Fig. 3 showed, in the different amount of precipitation
event, the results are consistent in all event, the peak discharge
in No.10 evergreen coniferous is higher than in No.12
deciduous broadleaf. However, the discharge in No.10
evergreen coniferous faster decline after reach the peakcompared to discharge in No.12 deciduous broadleaf. In Fig.
4, clearly seen snow depth in No.12 deciduous broadleaf isdeeper than in No.10 evergreen coniferous.

INTRODUCTION
In Japan after World War II many natural forest was change
to coniferous plantation forest for timber production. The
plantations were dominantly formed by converting native
broadleaf forest to coniferous forest (Hirata et al., 2006).
Converting vegetation type have big impact on hydrologicalcycle in watershed scale (Bosch and Hewlett, 1982, Chen etal., 2009). In Japan, forest covers most mountainous region
located upstream of agriculture and urban areas. From
viewpoint of water resource management, the forested areas
are considered to be water sources (Sawano et al., 2005;
Kumagai et al., 2014) and are intimately linked to downstream
ecosystems (Gomi et al., 2009). Converting broadleaf forest to
coniferous forest may influence the hydrological condition,
where this condition must be considered by water resource
management to make policy in future.
The objectives of this study is to compare the hydrologicalcharacteristic between deciduous broadleaf and evergreen
coniferous forest. The results of this study will provide useful
information for water resource management in such areas.

DISCUSSION
The result showed that the annual runoff from theconiferous evergreen forest was smaller than that from thebroadleaf deciduous forest (Fig. 1). One of the reasons of thisdifference in the annual runoff can be the phenologicaldifference between broadleaf deciduous and coniferous
evergreen forests. While coniferous evergreen forests always
keep leaves, broadleaf deciduous forests lose almost all leaves
in autumn. Due to no leaves in broadleaf deciduous forests
during winter season, the more snow can reach the ground. In
contrast, coniferous evergreen forests intercept a lot of snow
by canopy, and thus, the less amount of snow reaches theground (Fig. 4). The highest interception for the broadleaf
deciduous forest was 29% in UK (Herbst et al., 2008), whilethat for the coniferous evergreen forest was 45% in Sweden(Alavi et al., 2001). Leaf shape and configuration affect LAI
and water storage (Jonckheere et al., 2004, Keim et al., 2006).
Some leaves only store water as a thin coating whilst others
also store in capillary spaces between leaves. For thesereasons, flat leaves (deciduous species) store water less thantrees with needle leaves type (coniferous species) (Keim et al.,2006). Water storage by mature deciduous with and without
leaves is 0.2-2 mm and 0.03-0.8 mm respectively (Leyton etal., 1967), and 0.1-4.3 mm for mature coniferous tree (Link etal., 2004). Thus, if we can assume that annual interception ofboth forest types is comparable, annual ET of coniferous
evergreen forest is larger than that of broadleaf deciduous
forest. Then, under the same climatic conditions, we canconclude that annual runoff from coniferous evergreen forests
is smaller than that of broadleaf deciduous forests. For shortterm runoff (based on rainfall event) characteristics between
different forest types, found that the discharge in a coniferous
evergreen forest was always higher than that in a broadleaf
deciduous forest regardless of the magnitudes of storm events:a small autumn rainfall, a middle typhoon storm, and a large
typhoon storm.
Cypress plantations usually exhibit overstocked stands
having sparse or no understory vegetation cover due to the low
light conditions (Onda et al., 2010). The similar phenomenon
was observed in this research about little or no understory
vegetation cover in coniferous forest. On the other hands,
shows a high dense understory vegetation cover in a broadleaf
deciduous forest. Consequently, poor litter layers or nounderstory vegetation which were strongly affected by

MATERIALS AND METHODSThe Kuraiyama experimental forest is located in Gero City,
Gifu prefecture, Japan, between longitude 137°11´-137°14´ E
and latitude 35°58´-36°01´ N. Elevation is between 8201451m). The investigation carried out in No.10 evergreen
coniferous and No.12 deciduous broadleaf. There is a right
triangle weir has been built at each stream of basin and datalogger has been placed at the bottom of stream. The
precipitation data collected using tipping bucket rain gauge,
has been placed in No.12 deciduous broadleaf basin.
The No.10 basin forest is an evergreen coniferous forest of
Chamaecyparis obtuse (dominant tree species). This basin
located in the south of experimental forest, elevation between
926-1278 m and area is 0.6 km2. 74% of the basin is covered
by 40-50 years old artificial coniferous forest, 18% arebroadleaf forest and 8% are a natural coniferous forest.
The No.12 basin forest is a deciduous broadleaf forest of
Quercus sp (dominant tree species), the area is at the south of
No.10 basin area. Elevation between 909-1278 m, area is 0.73
km2. 77% of the No.12 basin is covered by deciduous
broadleaf forest, 14% is covered by 50-70 years old artificial
coniferous forest, and 9% is covered by natural coniferous
forest. The ground is also covered with a high density of bush,sasa bamboo grass, and litter layer.

RESULTSThe flow duration curve (FDC) is a plot is a plot shows thepercentage of tie that flow in a stream is likely to equal or
exceed some specific value of interest. FDC is one way tolook water discharge characters. Based on Fig.1, FDC
showed, more than 95% daily discharge in No.12 deciduous
broadleaf is higher than in No.10 evergreen coniferous.
However, in Fig.1 clearly seen around 2% the discharge in
No.10 evergreen coniferous is higher than No.12 deciduous
broadleaf.

96Proceedings of International Symposium on Animal Production and Conservation for Sustainable Development 2018
UGSAS-GU & BWEL Joint Poster Session on Agricultural and Basin Water Environmental Sciences 2018: P08

coniferous evergreen forest induced high peak discharge andhigh surface runoff compared with the broadleaf deciduoussite. Sakai et al. (2009) also indicated that a litter layer had
become very sparse and soil surface was exposed at bothcypress and cedar plots, while the deciduous plot was coveredby a thick litter layer and understory vegetation. The existenceof the understory vegetation cover can delay the speed ofsurface runoff and reduce peak discharge in broadleaf
deciduous forests. Sakai et al. (2009) and Hirano et al. (2009)
suggested that surface runoff at the cypress and the cedar plot
was a root flow (shallow flow through the root layer becauseof no litter layer) and that the surface runoff at the broadleaf
deciduous plot was a litter flow due to its thick litter layer.

REFERENCES
Hirata, Y., Matsumoto, M., and Iehara, T., (2006) Japanesenational forest inventory and its spatial extension by remotesensing. Proceeding of the eighth annual forest inventory
and analysis symposium. 16-19 October 2006. Monterey,
California. USA.
Bosch, JM., and Hewlett, JD., (1982) A review of catchment
experiments to determine the effect of vegetation changes
on water yield and evapotranspiration. J. of Hydrol., 55: 323.
Chen, Y., Xu, Y., and Yin, Y. (2009) Impacts of land usechange scenarios on storm-runoff generation in Xitiaoxi
basin, China. Quat. Int., 208:121-128.
Kumagai, T., Tateishi, M., Miyazawa, Y., Kobayashi, M.,
Yoshifuji, N., Komatsu, H., Shimizu, T. (2014) Estimationof annual forest evapotranspiration from a coniferous
plantation watershed in Japan (1): Water use components inJapanese cedar stands. Journal of Hydrology. 508: 66-76.

.

Fig.1 Flow duration curve from 2008-2017
a

b

c

d

Fig.2 Peak discharge (a), direct runoff (b), runoff coefficient (c), relationship rainfall with direct runoff (d) in No.12 deciduous
broadleaf and No.10 evergreen coniferous

Fig.3 Hydrograph No.12 deciduous broadleaf and No.10 evergreen
coniferous (up: 100 mm/event, down: 35 mm/event)97

Fig.4 Snow depth in No.12 deciduous broadleaf
and No.10 evergreen coniferous

Proceedings of International Symposium on Animal Production and Conservation for Sustainable Development 2018
UGSAS-GU & BWEL Joint Poster Session on Agricultural and Basin Water Environmental Sciences 2018: P09

Changes of pathogenic bacterial indicators during vermicompostingtreatment of sewage sludge
Shuailei Li1, Guangyu Cui2 and Fusheng Li3
1. Graduate School of Natural Science and Technology, Gifu University, Japan
2. Graduate School of Engineering, Gifu University, Japan
3. River Basin Research Center, Gifu University, Japan
Microbial analysis—Total coliform and E. coli wereanalyzed by plate count method. In brief, 2g of centrifugal
INTRODUCTION
sludge was added to an 18 mL tube containing physiologicalsaline solution and oscillation was performed for 30 minutes at
Sewage sludge is not only rich in nutrients, but also
200 rpm for distribution of bacteria uniformly in the tube.
contains a large number of pathogenic microorganisms.
1mL of the solution was taken and injected into another tube
Improper treatment and disposal will carry pathogenic
containing 9 ml saline solution for dilution. This process waspathogens into the environment, causing disease transmission,
repeated in the same way till 102, 103,104 folds’ dilutions werethus seriously threatening public health and ecologicalachieved. 1mL of the sample was poured to a Petri dishes
security. Vermicomposting is an environmentally friendly andcontaining Chromocult® Coliform Ager (Merk KGAA,
sustainable urban sludge treatment technology. In particular, it
Darmstad, Germany) and incubated for 24h at 37䉝㻚㻌㻭㼘㼘㻌㼐㼍㼞㼗is an effective management tool for organic solid waste forblue to violet colonies were counted as E. coli, and all salmon
developing countries owing to its low operational cost andto red colonies were counted as total coliform. Fecal coliforms
high application value for generation of vermicompost thatwere analyzed by M-FC method.
can be used as fertilizers or soil modifiers.
Despite numerous studies (Monroy et al., 2009; HenaultStatistical analysis— Statistical analysis was done using
Ethier et al., 2016) conducted on pathogenic bacteria survival
SPSS 19.0. One-way analysis of variance (ANOVA) was usedin vermicompost, a better understanding of the mechanisms
to test the significance of observed differences over time.
involved is still necessary. One of the major obstacles to the
Principal component analysis (PCA) was conducted based onwidespread acceptance of vermicomposting as a general
CANOCO 5.0.
organic solid waste management alternative is the insufficient
removal potential of pathogenic microorganisms duringvermicomposting (Edwards et al., 2010)
Therefore, to
RESULTS AND DISCUSSION
study the behavior and elimination mechanism of pathogenic
bacteria in the process of sludge treatment can not only
provide risk assessment for the treatment of sewage sludge by
Pathogenic bacterial indicators—The Fig. 1, Fig. 2, and Fig.
earthworms, but also improve the efficiency of sludge
3 showed the influence of earthworms on total coliforms, fecal
sanitation and safety utilization. The goal of the present study
coliforms and E. coli respectively.
is to evaluate the changes of total coliforms, fecal coliforms
1.6E+07and E. coli during the treatment of sewage sludge under
different density of earthworms and its possible mechanism.
1.4E+07
TC-C

MATERIALS AND METHODS
cfu/g dry weight

1.2E+07

Return activated sludge—Returned activated sludge wascollected from the North Wastewater Treatment Plant of Gifucity, Japan. It was used as the test sludge after sedimentationand centrifugation.

TC-20
TC-40

1.0E+07TC-60
8.0E+06
6.0E+06
4.0E+06

Earthworm—Earthworm used in this study is Eisenia

2.0E+06

foetida and the average weight is 0.30g. The earthworms had
been fed with sewage sludge for six months in the laboratory.

0.0E+00

0

5

10

15

Day

20

25

30

35

Fig. 1 Changes of total coliform duringvermicompostingExperimental set up— The experiment was designed forthree groups. Each reactor was loaded with 500 g of sludge,
and 20, 40 and 60 earthworms were introduced into the reactor
respectively (referred here after as E20, E40 and E60). In
addition, a control group E0, referred to the reactor added with
500g sludge but without earthworms, was also designed. The
reactor was placed in a constant temperature chamber at 25䉝㻌and sampled once a week for 31 days.

As shown in Fig. 1, the number of total coliforms incompost products decreases with the change of compostingtime. A week later, it showed a slow downward trend.
Compared with the control group, under the condition of highearthworm density, the total number of coliforms was higher
than that of the low density group. The removal rate of total
coliforms can reach 86%.

Physicochemical parameter— Water content, organic
matter (OM), dehydrogenase activity (DHA) of the wet
samples collected during composting were determined,
together with pH, electrical conductivity (EC), dissolved
organic carbon (DOC) and C/N ratio using the samples after
drying.

98

Proceedings of International Symposium on Animal Production and Conservation for Sustainable Development 2018
UGSAS-GU & BWEL Joint Poster Session on Agricultural and Basin Water Environmental Sciences 2018: P09

60

2.5E+06
FC-C

50

FC-20

40

E0
E20

DOC (g/kg )E40

2.0E+06

cfu/g dry weight

FC-40
FC-60

1.5E+0630
20
10

1.0E+0600

5.0E+05

7

14
Day

21

31

Fig. 5 Variations of DOC during the vermicomposting
Compared with the initial sludge (78.14%), the organic
matter in the final compost product (68.86%) decreased byabout 10%. The higher the density of earthworm, the faster theorganic matter will decline. During the treatment, DOC
decreased gradually. The higher the density of earthworm, themore the DOC concentration will drop.

0.0E+00
0

5

10

15

20

25

30

35

Day

Fig. 2 Changes of fecal coliforms during vermicomposting
The number of fecal coliforms in E0, E20 and E40 groups
decreased gradually during composting. The number of fecal
coliforms in the group of E60 increased abruptly on theseventh day, then decreased gradually.

Correlation analysis— Correlation analysis were conductedby SPSS 19.0 and CANOCO 5.0. The Fig. 6 showed theresults of redundancy analysis (RDA). The abundance of thepathogenic bacterial indicators had a strong positive linear
correlation with OM, DHA and DOC that could be probably
linked to such nutrients as labile C and labile sugars.

1.4E+06
EC-C

1.2E+06

EC-20

cfu/g dry weight

E60

1.0E+06

EC-40
EC-60

8.0E+05
6.0E+05
4.0E+05
2.0E+05
0.0E+00
0

5

10

15

Day

20

25

30

35

Fig. 3 Changes of E. coli during vermicomposting
As Fig. 3 shows the number of E. coli decreased graduallyduring the process. Compared with the changing trend of fecal
coliforms shown in Fig. 2, the changing trend of the number
concentration of E. coli was consistent with fecal coliform.

Fig. 6 Redundancy analysis

In the experiment, we found that the density of earthworm
doesn’t play a noticeable role in the reduction of pathogenic
bacteria. The abundance of the pathogenic bacterial indicators
had a strong correlation with OM and DOC that could be
probably linked to nutrients in the substrate. Research toclarify the relation between pathogenic bacteria and nutrient
should be carried out.

Chemical properties—As shown in the Fig. 4 and Fig. 5,variation of OM (Organic Matter) and DOC (Dissolved
Organic Carbon) reflected the changes of nutrients during thevermicomposting. It also indicated that the vermicompost
tended to stabilization.
80

OM(%)

REFERENCES

E0
E20
E40
E60

77
74

Edwards C.A., Subler S. (2010) Vermiculture Technology:
Earthworms, Organic Wastes, and Environmental
Management. Florida: CRC Press, 249-261.
Henault-Ethier L., Martin V.J.J., Gelinas Y. (2016)
Persistence of Escherichia coli in batch and continuousvermicomposting systems. Waste Management 56: 88-99.
Monroy F., Aira M., Dominguez J. (2009) Reduction of total
coliform numbers during vermicomposting is caused byshort-term direct effects of earthworms on microorganisms
and depends on the dose of application of pig slurry.
Science and Total Environment 407: 5411-5416.

71
68
65
0

7

14
Day

21

31

Fig. 4 Variation of organic matter duringvermicomposting99

Proceedings of International Symposium on Animal Production and Conservation for Sustainable Development 2018
UGSAS-GU & BWEL Joint Poster Session on Agricultural and Basin Water Environmental Sciences 2018: P10

Estimation experiment of complex terrain atmospheric structure
coefficient ࡯૛࢔ by weather forecast model
Yuto Ito1, Tomonao Kobayashi2 and Jun Yoshino2
1. Graduate School of Natural Science and Technology, Gifu University, Japan
2. Faculty of Engineering, Gifu University, Japanconsidered from optical and meteorology perspectives. This
joint observation was also carried out at Shonan campus of
Tokai University in Hiratsuka city, Kanagawa prefecture, in
September 2017 and March 2018. In this research, I’llestimate ‫ܥ‬௡ଶ by weather forecast model for the joint
observation in December 2016 and March 2018, and its result
is compared with the observation result. In two joint
observation points, altitude and land use are markedly
different. So I’ll compare and consider about the influence of
this terrain on ‫ܥ‬௡ଶ estimation by numerical model.

INTRODUCTION
Recently, the propagation of laser is becoming more
important for transmission system realization of mass data. A
barometer of the degree of a disturbance of atmosphere that
influences on the propagation of laser is ‫ܥ‬௡ଶ . However theappropriate model for estimation has not been drawn up. Then
to make the appropriate model for estimation of ‫ܥ‬௡ଶ when
you set the certain weather conditions and environmental
conditions is a problem for transmission system realization.
In this research, I’ll estimate ‫ܥ‬௡ଶ express a disturbance of
atmosphere by using weather forecast model (WRF). And I’ll
give a possibility of the numerical model to compare themodel and observation.

(2)
The influence of terrain condition on ࡯૛࢔ estimation
Figure1 and 2 shows that altitude and land use distribution of
Taiki town, and Hiratsuka city used in numerical calculation
model by WRF Domain2 to discuss influence of terrain
conditions. In figure1,2 target point is (50,50). Grid size is
1km.Figure3.4 shows that correlation between result of ‫ܥ‬௡ଶ
estimation and result of ‫ܥ‬௡ଶ observation. As shown in Fig.1,
there is a sea in the southeast direction, but the target area isflat and land use is almost uniform. So as shown in Fig.3 that
reproducibility of ‫ܥ‬௡ଶ estimation is high. But as shown in
Fig.2 that there are somewhat ups and downs, especially land

MATERIALS AND METHODS(1)
Analytical method and term
Joint observation was conducted by some companies on a
disturbance of atmosphere of the laser propagation at JAXATaiki aerospace experiment station in Taiki town, Hokkaido in
December 2016. This disturbance of atmosphere wasFig.2 altitude and land use distribution of Hiratsuka city,

Fig.1 altitude and land use distribution of Taiki town,
Hokkaido㸦JAXA Taiki aerospace experiment station㸧

Kanagawa prefecture㸦Shonan campus of Tokai university㸧

100Proceedings of International Symposium on Animal Production and Conservation for Sustainable Development 2018
UGSAS-GU & BWEL Joint Poster Session on Agricultural and Basin Water Environmental Sciences 2018: P10

use is complicated around Tokai university Shonan campus. Ina smaller scale, there are many lecture buildings and high trees
in campus and you can easily imagine that an air current issmall and complicated that can not be reproduced by WRF. So
as shown in Fig.4 shows that reproducibility of ‫ܥ‬௡ଶ
estimation by WRF is low. According to these results,
accuracy of ‫ܥ‬௡ଶ estimation is influenced by topography andland use. In an atmospheric turbulence field, Monin-Obukhov
similarity can usually be applied as a turbulent structure. The
influence of the ground surface has a significant influence on
the atmosphere and disturbance within the atmosphereboundary layer close to the ground surface targeted in thisstudy. ‫்ܥ‬ଶ and ‫ܥ‬௡ଶ are calculated by sensible heat flux andfriction speed estimated using WRF by similarity of MoninObukhov in the ground boundary layer.

RESULTS AND DISCUSSION(1)
Result of analysis
From Fig.3, some data of ‫ܥ‬௡ଶ estimation by WRF isunderestimated compared to the ‫ܥ‬௡ଶ observation. We knowthat this underestimated period is the nighttime zone. WRF
estimates that the sensible heat transport is small andatmosphere is stable. However there are actual temperaturefluctuations. In other periods, the ‫ܥ‬௡ଶ estimation by WRF ishighly reproducible for the ‫ܥ‬௡ଶ observation.In this figure, ‫ܥ‬௡ଶ
estimation by WRF is smaller compared to ‫ܥ‬௡ଶ observation
sunset to sunrise. In this figure, ‫ܥ‬௡ଶ estimation in daytime. On
the other hand, estimation is bigger than observation innighttime. Moreover, the plot is spread widely as a whole, thecorrelation between ‫ܥ‬௡ଶ estimation and ‫ܥ‬௡ଶ observation islow, and the estimation accuracy of WRF seems to be low.
However, if you distinguish daytime and nighttime,correlations can be seen.

Fig.4 Correlation between result of ‫ܥ‬௡ଶ estimation andresult of ‫ܥ‬௡ଶ observation 㸦 Shonan campus of Tokai
university㸧

CONCLUSION
In this research, we reproduced the state of the atmosphere
for joint observation of Taiki town in December 2016 andHiratsuka city in March 2018 by WRF. So we estimated ‫ܥ‬௡ଶ .
Furthermore we compared its result with observation. The
reproducibility of estimation is influenced by altitude and land
use.

REFERENCES
Tamagawa, I., T. Kanazawa, H. Yoshida, Y. Takayama, A.
Hayakawa: Comparing ‫ܥ‬௡ଶ evaluation by weather
observation and ‫ܥ‬௡ଶ evaluation by scintillometer,
61st lecture meeting of space science union skill , 2A09,
2017.
Yoshida, H., Y. Takayama, C. Huzikawa, T. Kobayashi, J.
Yoshino, I. Tamagawa: Observation for influence of
weather and atmosphere on the propagation of laser, 62st
lecture meeting of space science union skill, 1G01, 2018.
Tamagawa, I., T. Kobayashi, J. Yoshino, Y. Takayama, C.
Huzikawa, Y. Yoshida, T. Suzuki, R. Kuribayashi, K.
Kitakura: ‫ܥ‬௡ଶ estimation by weather observation, 62st
lecture meeting of space science union skill, 1G02, 2018.
Yoshino, J., T. Kobayashi, I. Tamagawa, H. Yoshida, Y.
Takayama, C. Huzikawa, T. Suzuki, R. Kuribayashi, K.
Kitakura: 62st lecture meeting of space science union skill,
1G03, 2018.
Kobayashi, T., I. Tamagawa, J. Yoshino, H. Yoshida, H.
Yoshida, Y. Takayama, A. Hayakawa: Estimation
experiment ‫ܥ‬௡ଶ by weather forecast model, 2A10, 2017.
WRF
Model
Users
Page:
http://www.mmm.ucar.edu/wrf/users/
Monin, A.S., Obukhov,A.M. : Osnovnye zakonomernosti
turbulentnogo peremcsivanija vprizemnom sloe atmosfory,
Trudy geofiz inst AN SSR, Vol. 24(151), pp.163-187.

Fig.3 Correlation between result of ‫ܥ‬௡ଶ estimation andresult of ‫ܥ‬௡ଶ observation㸦JAXA Taiki aerospace experimentstation㸧101Proceedings of International Symposium on Animal Production and Conservation for Sustainable Development 2018
UGSAS-GU & BWEL Joint Poster Session on Agricultural and Basin Water Environmental Sciences 2018: P11

Estimation of small hydropower generation using river discharge water inheadwork
Fenglan Wang¹, Masateru Senge ² and Keigo Noda ²
1. The United Graduate School of Agricultural Science, Gifu University¹
2. Faculty of Applied Biological Sciences, Gifu University, Japan²(1997~2017) is used and for INUYAMA headwork, 10 years’
data (2008~2017) is used.
The generation output(kW) was calculated with using the㸻 g × Q × 㹆îȘ
form: P㸻
(p: generation output[kW], g: gravitational acceleration(9.8) [m/s²], Q: flow rate[m³/s@ +GURSȘJHQHUDOHIILFLHQF\[0.72㸻0.825(water wheel efficiency) × 0.875(generator
efficiency)].
For the calculation process, firstly, the excess probability (isshown as percentage, to make the daily flow rate of headwork
in descending order, then to divide that rank by the total
number of days to change them to percentage). Secondly, theutilization rate was calculated when the flow rate
corresponding to excess probability 10% to 90% was taken as
the maximum water usage. Thirdly, the relations between
maximum output and utilization rate is estimated. Fourthly,
the output is obtained when the utilization rate is 60% and it isused as maximum output in the calculation. Last, the actual
annual and monthly generation capacity are calculated withusing maximum output when the utilization rate is 60%. The
usage rate of small-hydropower power generation equipment
is estimated to be at least 60% as a standard forcommercialization according to Energy · Environment
Conference Cost etc. Verification Committee in Japan. Some
conceptions are related in this paper. Here, I will introduce
these conceptions. Annual generation capacity: annual
generation amount when using the maximum water usage(maximum water usage: the maximum value of the flow ratethat a facility can use). Actual generation capacity: annual
generation amount when using actual water usage (actual
water usage: actual flow rate with maximum water usage as
upper limit). Utilization rate (%): division of actual annual
generation capacity and annual generation capacity.

INTRODUCTION
In recent years, receiving the influence of action for
reduction of greenhouse gas(CO΃) and accident at Tokyo
Electric Power Company's Fukushima No. 1 nuclear power
plantˈ people began to focus on renewable energy such as
solar, wind power, hydropower, geothermal, biomass andwave power. We have great renewable energy recourse to
make generation. It is purely domestic energy that we do notneed to import the energy sources from other countries.
It has been worked eagerly for promotion of introductionof renewable energy use. Thus some strategies or policies
were taken by government, like Feed-in-tariff law, taking
2002 as the beginning year to develop the renewable energy
and etc. Tariff price of small hydropower (SHP) generation,
35.7 yen for 1 kWh when the output is below 200kW, 30.45
yen when the output is from 200kW~1000kW for 1 kWh and25.2 yen when the output is from 1000kW~30000kW for
1kWh (Agency for Natural Resources and Energy, 2002), also
encourage the diffusion of SHP generation.
There is no clear definition of small hydropower in Japan.
However, according to the output, it is classified as largehydropower, middle hydropower, small hydropower, minihydropower and micro hydropower. In this research, we referfacilities with output less than 1000kW to small hydropower.
SHP generation is a kind of distributed energy and facility
utilization rate is higher in the renewable energies.
In Japan, it has great potential SHP generation, and there
exists many unused falling heights to be utilized for making
SHP generation. For SHP generation using agricultural watersupply facilities, utilization of adjustment reservoir anddistribution water to make generation has been planning andcarrying out rapidly in recent years.
Headwork is one of agricultural irrigation facilities
developed since ancient times to intake the agricultural waterfrom the rivers.
In this research, we focus on the discharge water from the
upstream part to the downstream part of headwork to evaluate
the generation potential with using its’ falling highs and thisdischarge water. We would like to evaluate the possibility ofgeneration utilizing the discharge water of headwork byanalysis of annual generation and monthly generation

RESULTSFor the results of this thesis, I will introduce the results of
one site-MEIJI YOUSUI headwork as an example.
Figure1 shows the excess probability. Figures 2 shows therelation between maximum output and utilization rate, and we
can get the output when the utilization rate is 60% from thisfigure. Then, we set this output as maximum output andobtained the annual and monthly generation capacity (Figure 3
and Figure 4). For MEIJI YOUSUI headwork, the maximum
output is 947 and we got the annual generation capacity from3196~6609MWH (average annual generation is 4971MWh)and average monthly generation is 414 MWh, the biggest
generation is 577MWh in October and 303MWh in June. It
obtained the relatively stable generation through the year.

MATERIALS AND METHODS
There are total 8 sites in Gifu prefecture and Aichi
prefecture are estimated totally. They are in ONYU headwork,
FURIKUSA
headwork,
KANSAGAWA
headwork,
MUROMATSUBARA headwork, HOSOGAWA headwork,
OKAJIMA headwork, INUYAMA headwork and MEIJI
YOUSUI headwork.
We plan to use the sixteen years’ data of flow rate
(2002~2017) to estimate the generation capacity. Amongthem, for MEIJI YOUSUI headwork, 21 years’ data102

Proceedings of International Symposium on Animal Production and Conservation for Sustainable Development 2018
UGSAS-GU & BWEL Joint Poster Session on Agricultural and Basin Water Environmental Sciences 2018: P11

Until now, we finished the calculation of potential generation
for seven sites. And it is made a conclusion in a table
Table1 annual and monthly generation capacity for seven research sites.

REFERENCES
Ministry of Economy, Trade and Industry, Agency for Natural
Resources and Energy · Japan Energy Foundation: Hydro
Valley Planning Guidebook, 2005:3-4.

DISCUSSION
Until now, we have finished the calculation ofpotential generation for seven sights. From the present
results, From the present results, we got the relatively
big potential generation and quite stable monthly
generation capacity. The annual average actual
generation we got totally is 18976MWh for 5 researchsites. It can provide for 2447 person’s annual electricity(In Japan, annual electricity consumption of per person
is 7753kWh.
To complete this thesis, it needs to clarify some waterrights problems related in using the discharge water inheadwork. And also, the basin condition (such as: theresource of water in headwork and where to be flowninto) each headwork needs to be evaluated clearly.

Tanaka Satoru (1987) Improvement of Irrigation and Drainage
System. Japanese Society of Irrigation, Drainage andRural Engineering, 55: 5-10
Energy · Environmental Conference Cost etc. Verification
Committee: Report on Cost etc. Verification Committee,
2011,p.57
Ministry of Agriculture, Forestry and Fisheries Rural
Promotion Bureau: Promotion of small hydropower
generation utilizing agricultural irrigation facilities, 2014,
p.1-12.

103

Proceedings of International Symposium on Animal Production and Conservation for Sustainable Development 2018
UGSAS-GU & BWEL Joint Poster Session on Agricultural and Basin Water Environmental Sciences 2018: P12

A study on Kotsu-yousui area regional water requirement for paddy fieldby estimating the repeated use of water field
Suozhu1, Masateru Senge2 and Keigo Noda2
1. The United Graduate School of Agricultural Science, Gifu University
2. Faculty of Applied Biological Sciences, Gifu University, Japanactual situation of the water supply management irrigation
requirement in the district that could not evaluate impact
INTRODUCTION
statement of the possibility of water quantity in the district.
And, we aim to estimate the actual condition of distribution of
In recent years, the agricultural land area in Japan tends tomanagement water.
be decreasing due to the increase of general-purpose fields, thereduction of repetitive use due to separation of irrigation anddrainage canal, the aging of agricultural workers, urbanizationand etc. Based on this background, we choose the Kotsuyousui as the research area, at first, we will reorganize theirrigation and drainage system in accordance with the current
gross area and irrigation and drainage system, then we
estimate the quantity of planned water intake based on the CB
method using a new irrigation and drainage system, aiming tograsp the present water demand.
㻷 㻻㼀㻿㼁㻙㼅㻻㼁㻿㼁㻵㻳㻻㻶㼅㻻㻌㻷㻭㼃㻭㻿㻴㻵㻺㻌㻷㻻㼀㻿㼁㻙㼅㻻㼁㻿㼁㻵

12

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MATERIALS AND METHODS

42

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The First of all, we will give an explanation of geographicalcondition and water condition of Kotsu-Yousui area which is
the investigation site of this research. The field survey andanalysis were conducted to reorganize the irrigation anddrainage canal. According to the field survey, we grasped the
profit paddy field area in the target field survey area andconfirmed the irrigation and drainage canal in the paddy field,
and made new blocks.
In order to grasp the amount of required water, we
conducted a survey to measure the flow rate of rice paddy
canal. We selected 28 observation points of important riverand irrigation canal in three water type IWAKURA Yousui,
Aisekawa Yousui, Shinkostu Yousui in Kotsu-yousui Area,
We measured the actual flow rate for five times in 2015
according to the regional water management.
The CB method is a method created by improving both of
the water requirement in depth and water balance method.
While it is considering the reduction and repetitive use within
the wide area of paddy filed, when it can be assumed as theflowing water in irrigation and drainage canal is near to themost steady state, that is, we planned to estimate themaximum required water amount in the period of continuous
dry weather in the training period.
In order to investigate the maximum requirement wateramount, area of paddy field in 2016 was calculated based on
the map and the field survey, and the transition (changes) of
paddy field area and building area were compared in 1978,
1990 and 2016.
According to the survey and research results, the decreaseof agricultural land and the increase of building land wereobserved in Kotsu-yousui area, and the change of land use
accompanying with urbanization became clear. In particular,
in Kita Nagoya city, paddy field area decreased remarkably.
As a result, it was found that when the maximum required
amount is estimated by using the CB method, the actual water
intake is excessive.

85

56

80

46

30
79

86

57
31

47

58
59
34

37

41

48

87
60
32

35

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89

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91

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83

82

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㼕㼞㼞㼕㼓㼍㼠㼕㼛㼚㻌㼏㼍㼚㼚㼍㼘㼐㼞㼍㼕㼚㼍㼓㼑

52㻺㻮㻌㻮㼘㼛㼏㼗
P

㻼㼡㼙㼜Fig. 1 Blocks of Kotsu -yousui area (1978).

DISCUSSION
In this study, we measured the amount of water intake in
Kotsu-yousui area and proposed a decision result. I want tocontinue this study in order to make more accurate judgment.
In the Kotsu-yousui area there are many rivers, the main water
system joins together with the river and we will estimate thequantity of the final drainage water and evaluate the influenceof the final drainage water of rice paddy on the repetitive use
structure of agricultural water in the wide paddy field of
Kotsu-yousui, in combination with the quantity water intake.

REFERENCES
Mizutani M, Hirota T and Kimoto Y (1980) A study on theplanning of seasonal water requirement for paddy field by
estimating the repeated use of water. The Bulletin of theFaculty of Agriculture, Mie University. 61:97-228.
Yoshida M, Maruyama T, Noto F, Takase K and Takimoto H(2012) Analysis of return flow of irrigation water at test
paddy in the Tedori River alluvial fan area. IDRE Journal,
282: 35-42.
Okamoto M and Satho M (1990) Some characteristics ofirrigation for paddy in Japan from the viewpoint of

RESULTSIn Kotsu-yosui district, we plan to use the CB method which isa calculation method of the irrigation requirement inconsideration of the repetition use, and aims to grasp the104Proceedings of International Symposium on Animal Production and Conservation for Sustainable Development 2018
UGSAS-GU & BWEL Joint Poster Session on Agricultural and Basin Water Environmental Sciences 2018: P12hydrology and water resources engineering. Japan society
of hydrology and water resources, 31: 1-6.
Satoh M and Okamoto M (1985) Theoretical study of block
type classification in the critical block method. Trans.
JSIDRE, 118: 17-22.
Senge M, Nishide T, Ohta A and Ogura K (1995) Analysis ofeffective rainfall in paddy field district by applying CB
Method in rainy days. Trans. of JSIDRE, 180: 21-28.
Nakagiri T, Ando H, Hirayama S, Ishikawa S and Maruyama
T (1999) Management of irrigation water in extraordinary
drought conditions ̽Case study of 1994 drought in theWest Japan㸫. J.Japan Soc. Hydrol. & Water Resour. 12(3):242-249.
Sawada S, Kogawa H and Sato D (1999) Estimation of humanactivity affecting water balance of the Iwaki River basin,
Society Environmental Science, Japan 12(2):171-183.
Nakagiri T, Watanabe T, Horino H and Maruyama T (2000)
Analysis of sufficiency and reuse of irrigation water in the.LQR 5LYHU %DVLQ ʊ $QDO\VLV RI LUULJDWLRQ ZDWHU XVH E\ D
%DVLQ +\GURORJLFDO 0RGHO ,,  ʊ  7UDQV RI -6,'5(
205:35-42.
Kotsu-yousui
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Fig2. Blocks of Kotsu-yousui area (2018).

105

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HabashitaProceedings of International Symposium on Animal Production and Conservation for Sustainable Development 2018
UGSAS-GU & BWEL Joint Poster Session on Agricultural and Basin Water Environmental Sciences 2018: P13

Recombinant ovine angiotensinogen as a promising substrate formeasuring renin concentration
Jobaida Akther1,2, A. H. M. Nurun Nabi2, Satoshi Ohno3, Takashi Yokogawa3,4, Tsutomu Nakagawa5, Fumiaki Suzuki5 andAkio Ebihara4,5
1. The United Graduate School of Agricultural Science, Gifu University, 1-1 Yanagido, Gifu 501-1193, Japan
2. Department of Biochemistry and Molecular Biology, University of Dhaka, Dhaka 1000, Bangladesh
3. Faculty of Engineering, Gifu University, 1-1 Yanagido, Gifu 501-1193, Japan
4. Center for Highly Advanced Integration of Nano and Life Sciences, Gifu University (G-CHAIN), 1-1 Yanagido, Gifu 5011193, Japan
5. Faculty of Applied Biological Sciences, Gifu University, 1-1 Yanagido, Gifu 501-1193, Japansubjected to SDS-PAGE using SuperSep Ace 12.5% gel, 13
wells (Wako, Tokyo, Japan). DSF experiments were
performed as described previously. To estimate the melting
temperature (Tm), the Boltzmann equation was used to fit thefluorescence data using GraphPad Prism 7.0 software(GraphPad Software, La Jolla, CA) and the calculation
templates. The Tm values were expressed as the mean ±standard deviation.

INTRODUCTION
Renin is the key enzyme of the renin–angiotensin–
aldosterone system (RAAS) which plays an important role incirculatory hemodynamics through its effects on vascular tone,
salt and water homeostasis [1,2]. This enzyme cleaves its
substrate, angiotensinogen (ANG), to produce the decapeptide
angiotensin I (Ang-I), which is subsequently converted into
angiotensin II inducing a vasoconstriction as well as
aldosterone release [1]. Plasma renin activity (PRA)
measurement got much importance as a substitute of RAAS
activity evaluation [3–5]. PRA was also found to be associatedwith increased risk of myocardial infarction, cardiovascularand all- cause mortality in hypertensive patients [6–9]. Now-adays direct renin assays that measure renin as massconcentration being faster, methodologically simpler andresults, being independent of ANG concentration areprogressively replacing traditional PRA assay [10]. Several
comparative studies showed that the reproducibility of plasma
renin concentration (PRC) is higher than that of PRA amonglaboratories [10]. PRC is defined in two approaches [11]. One
is plasma renin concentration measured by immunoassay (irPRC), which is expressed as mass concentration. The other isplasma renin concentration measured by activity assay (acPRC). ac-PRC is determined by measuring an enzymatic
activity of renin with an excess amount of renin substrate to
avoid the influence of variation in plasma ANG concentration[11].
Ovine ANG (oANG) has been shown as a better substrate tomeasure ac-PRC [11]. Assayed with human renin, oANG
shows 4.4× lower Michaelis constant (Km) and 2.1× higher
turnover number (kcat) than human ANG, thereby leading toabout 9× higher catalytic efficiency (kcat/Km) [12,13]. WhenoANG is utilized in ac-PRC measurement, a less amount of
ANG is needed to reach a saturating concentration of substrateowing to the lower Km.
We have recently reported a method of producing
recombinant oANG at a milligram level using E. coli
expression system [14]. Recombinant oANG produced by thismethod retains the similar activity and stability to that ofexpressed in mammalian cells. Since a large amount of oANG
is available now, we wish to establish a better clinical assay
for renin concentration measurement using our cost-effective
recombinant oANG.

Kinetic analysis- Various concentration (0.08 – 1.5 M) ofrecombinant oANG were incubated with recombinant humanrenin standard (27 pM) at 37°C for 30 min in 1× ES. The rateof Ang-I generation was determined by an enzyme-linked
immunosorbent assay (ELISA) [15]. The Km and maximum
velocity (Vmax) were estimated by Hanes-Woolf plot using
GraphPad Prism 7.0 software. The kcat of renin-ANG reaction
was calculated using the following formula:kcat = Vmax / renin concentration
Establishment of calibration curve of renin concentration
using recombinant oANG- Ang-I specific ELISA [15] wasused to measure the amount of Ang-I produced from various
concentrations of renin. Microplate Manager 6 Software (BioRad Laboratories, Inc., Hercules, CA, USA) was used to
calculate amount of Ang-I produced from the absorbance.
Enzymatic activity of renin (i.e., renin activity) is defined asfollows:
Renin activity = [pg amount of Ang-I / (0.1mL × 2 h
×1000)] ng Ang-I mL-1 h-1
Renin activity measurements were repeated six times (n= 6) to establish a calibration curve of renin concentration.

RESULTS
Production of recombinant oANG- Recombinant oANG was
expressed in E.coli cells and purified to homogeneity using
two steps of column chromatography (Fig. 1). The final
preparation contains oANG protein alone (lane 5, Fig. 1).
$ERXW  ȝ/ RI  ȝ0 UHFRPELQDQW R$1* ZDV REWDLQHG
from 0.5 L of E.coli cell culture, with a yield of about 9 mg
per liter of culture.
To examine the protein quality, the absorbance ofrecombinant oANG (1:20 diluted) was measured with a scan
range of 240-340 nm. The absorbance at 340 nm was almost
zero, indicating that there was no aggregation in the protein
preparation. Maximum absorbance was 0.562 at 279 nm andminimum absorbance was 0.015 at 340 nm.

MATERIALS AND METHODS
Production of oANG using E. coli cells- The production ofrecombinant oANG in E. coli was performed as described
previously [14].

Protein stability analysis of recombinant oANG- DSF wasused to examine the stability of recombinant oANG. The DSF
results showed that oANG undergoes a two-state transition
during the course of thermal denaturation. The Tm ofrecombinant oANG was 52.96 ± 0.22°C.

Physical properties of oANG- Fractions were pooled fromdifferent stages of recombinant oANG preparation and106Proceedings of International Symposium on Animal Production and Conservation for Sustainable Development 2018
UGSAS-GU & BWEL Joint Poster Session on Agricultural and Basin Water Environmental Sciences 2018: P13

results. With this recombinant oANG, picomolar amount ofrenin can be measured from known renin activity, whichshould be utilized in the clinical assay for measuring PRC.

Establishing calibration curve of renin concentration using
recombinant oANG- Various concentrations of renin in pM
range were prepared using recombinant human renin standard,
and the renin activities were measured at 0.8 M of oANG (5times higher than Km value). A linear calibration curve wasobtained by plotting the renin activity versus reninconcentration (0.2 – 1.5 pM), with a slope of 2.10, a yintercept of 0.02, and R2 of 0.99 (Fig. 2). This result
demonstrates a strong linear relationship between renin
activity and renin concentration. An intraassay CV of < 20%
was found in the six repeating experiments.

REFERENCES[1] C.M. Ferrario, Role of angiotensin II in cardiovascular disease
therapeutic implications of more than a century of research., J. Renin.
Angiotensin. Aldosterone. Syst. (2006).
[2] A.N. Nabi, K.B. Biswas, A. Ebihara, T. Nakagawa, F. Suzuki,
Renin Angiotensin System in the Context of Renin, Prorenin, and the(Pro)Renin Receptor, Rev. Agric. Sci. 1 (2013) 43–60.
[3] H. Wernze, H.J. Spech, G. Müller, Studies on the activity of therenin-angiotensin-aldosterone system (RAAS) in patients withcirrhosis of the liver, Klin. Wochenschr. 56 (1978) 389–397.
[4] E. Sulyok, M. Németh, I. Tényi, I. Csaba, E. Györy, T. Ertl, F.
Varga, Postantal development of renin-angiotensin- aldosterone
system, RAAS, in relation to electrolyte balance in premature infants,
Pediatr. Res. 13 (1979) 817–820.
[5] V.J. Dzau, W.S. Colucci, N.K. Hollenberg, G.H. Williams,
Relation of the renin-angiotensin-aldosterone system to clinical state
in congestive heart failure, Circulation. 63 (1981) 645–651.
[6] M.H. Alderman, S. Madhavan, W.L. Ooi, H. Cohen, J.E. Sealey,
J.H. Laragh, Association of the renin-sodium profile with the risk of
myocardial infarction in patients with hypertension, N. Engl. J. Med.
(1991).
[7] M.H. Alderman, W.L. Ooi, H. Cohen, S. Madhavan, J.E. Sealey,
J.H. Laragh, Plasma renin activity: a risk factor for myocardial
infarction in hypertensive patients, Am J Hypertens. (1997).
[8] J.D. Blumenfeld, J.E. Sealey, M.H. Alderman, H. Cohen, R.
Lappin, D.F. Catanzaro, J.H. Laragh, Plasma renin activity in theemergency department and its independent association with acute
myocardial infarction, Am J Hypertens. (2000).
[9] M.C. Gonzalez, H.W. Cohen, J.E. Sealey, J.H. Laragh, M.H.
Alderman, Enduring direct association of baseline plasma renin
activity with all-cause and cardiovascular mortality in hypertensive
patients, Am. J. Hypertens. (2011). [10] T. Meade, Review: Plasma
renin and the incidence of cardiovascular disease., J. Renin.
Angiotensin. Aldosterone. Syst. 11 (2010) 91–8.
[10] C. Lonati, N. Bassani, A. Gritti, E. Biganzoli, A. Morganti,
Measurement of plasma renin concentration instead of plasma renin
activity decreases the positive aldosterone-to-renin ratio tests intreated patients with essential hypertension, J. Hypertens. 32 (2014)
627–634.
[11] D.J. Campbell, J. Nussberger, M. Stowasser, A.H.J. Danser, A.
Morganti, E. Frandsen, J. Ménard, Activity assays and immunoassays
for plasma renin and prorenin: Information provided and precautions
necessary for accurate measurement, Clin. Chem. 55 (2009) 867–877.
[12] T. Goto, N. Imai, S. Hirose, K. Murakami, An improved methodfor determination of active and total renin concentration in humanplasma using an excess of sheep substrate, Clin. Chim. Acta. (1984).
[13] U.M. Nasir, K. Takahashi, T. Nagai, T. Nakagawa, F. Suzuki, Y.
Nakamura, Two Peaks in pH Dependence of Renin-angiotensinogenreaction, Biosci. Biotechnol. Biochem.
[14] S. Yamashita, N. Shibata, A. Boku-Ikeda, E. Abe, A. Inayama, T.
Yamaguchi, A. Higuma, K. Inagaki, T. Tsuyuzaki, S. Iwamoto, S.
Ohno, T. Yokogawa, K. Nishikawa, K.B. Biswas, A.H.M.N. Nabi, T.
Nakagawa, F. Suzuki, A. Ebihara, Escherichia coli-based productionof recombinant ovine angiotensinogen and its characterization as arenin substrate, BMC Biotechnol. 16 (2016).
[15] F. Suzuki, S. Yamashita, A. Takahashi, M. Ito, S. Miyazaki, Y.
Nagata, Y. Nakamura, Highly sensitive microplate-elisa forangiotensin i using 3,3’5,5’tetramethylbenzidine, Clin. Exp.
Hypertens. (1990).

Fig. 1. SDS-PAGE analysis of recombinant oANG. The stagesof oANG production were separated by SDS-PAGE andstained with CBB. Lane-1,6: molecular weight marker; Lane2: cell supernatant after sonication; Lane-3: after affinity
column chromatography; Lane-4: after ion-exchange column
chromatography; Lane-5: concentrated oANG after
SXULILFDWLRQ ȝJ 

Fig. 2. Renin concentrations vs. renin activity plot. Linear
regression analysis produced a slope of 2.10, a y-intercept of
0.02 and R2 of 0.99 (n = 6) suggesting linear relationshipbetween renin concentration and renin activity. Error bars areshowing standard error of mean values ranging from 0.02 0.07.

DISCUSSION

Renin concentration in plasma has significant prognostic value
in patients with cardiovascular and renal diseases. The sourceof renin substrate, ANG, to be used in the assay method forrenin evaluation is limited. This limitation can be solved using
recombinant oANG expressed in E. coli cells, based on our107Proceedings of International Symposium on Animal Production and Conservation for Sustainable Development 2018
UGSAS-GU & BWEL Joint Poster Session on Agricultural and Basin Water Environmental Sciences 2018: P14

Frequency of FUM21 mutation causing fumonisin non-producibility in
Fusarium fujikuroi strains
Sharmin Sultana1, Miha Kitajima2, Hironori Kobayashi2, Ryuou Yamaguchi2, Masafumi Shimizu2, Koji Kageyama3 andHaruhisa Suga4
1. The United Graduate School of Agricultural Science, Gifu University, Gifu 501-1193, Japan
2. Fac. of Applied Biological Sciences, Gifu University, Gifu
3. River Basin Research Center, Gifu University, Gifu, Japan
4. Life Science Research Center, Gifu University, Gifu 501-1193, JapanINTRODUCTION¾

Fumonisin is a kind of carcinogenic mycotoxin producedby 15 species of Fusarium fujikuroi species complex (Rheeder
et al. 2002). Fumonisin causes intestinal cancer and neuraltube defect in human and several disease in animals (Marasas
et al. 2004). F. proliferatum and F. fujikuroi are considered as
potential fumonisin producers on rice (Cruz et al. 2013). The
frequency and production level of fumonisin of F. fujikuroi
are partly attributed to different assay method and different
growth media used in individual investigation (Cruz et al.
2013, Bolton et al. 2016). On the basis of fumonisin
production we classified Fusarium fujikuroi into two groupsuch as F-group, produces fumonisin in significant level andG-group does not produce fumonisin in detectable level.
Fumonisin production is dependent on a biosynthetic gene
(FUM) cluster that composed of 16 genes (Proctor et al.
2003). The FUM cluster was detected not only from F-group
strains but also from G-group strains. FUM21, a Zn(II)-2Cys6
DNA binding transcription factor, positively regulates FUM
gene expression and is required for fumonisin biosynthesis(Brown et al. 2007). Without a functional FUM21 lacked
transcripts from two biosynthetic genes FUM1 and FUM8 andfailed to produce FB1 (Brown et al. 2007). It has been revealed
that low level of expression of FUM21 is the cause of low
fumonisin production in a F. fujikuroi strain (Rosler et al.
2016). Information of the genes affecting fumonsin
producibility can be a target for chemical development toinhibit this mycotoxin production. The information is also
important for accurate assessment of fumonisin production
risk of F. fujikuroi.
In previous study, genetic mapping using the crossing
progenies between a fumonisin producing strain Gfc0825009(G9) and a non-producing strain Gfc0801001 (G1) indicatedthat fumonisin non-production in G1 attributes to the FUM
cluster (໭ᔱ 2012). PCR results indicated that G1 retain
entire FUM cluster. Some important genes tested wereexpressed in G9 but only FUM21 and FUM1 expression weredetected in G1 in RT-PCR result. Though FUM21 expression
was detected in G1, lack of remaining three FUM gene
expressions suggested dysfunction of FUM21 in G1. We
identified the causative mutation in FUM21 and its
distribution in F. fujikuroi strains.

¾¾

RESULTS and DISCUSSION
RT-PCR of FUM genes
DNA with expected size of all genes tested were detected
in G9 by RT-PCR but it was detected only for HistoneH3,
FUM21 and FUM1 in G1 (Fig. 1A and 1B). Lack oftranscription of multiple FUM genes suggested dysfunction of
the transcription factor FUM21 in G1. Positive conversion

of FUM6, FUM8 and FUM10 expression by integrationof FUM21 was confirmed in a transformant
FfT21FUMKOD (#2) (Figure 1C).

Fig. 1: RT-PCR results of (A) G9, (B) G1 and (C)FfT21FUMKOD-2. (+) indicated the expected size of RTPCR product was detected, (-) not detected.
FUM gene complementation by transformation
Fumonisin production recovery was failed bycomplementation of only FUM21 but it was succeeded by
FUM6/ FUM7/ FUM8/ FUM10/ FUM11/ FUM2/ FUM13 (ᑠᯘ 2013, FfDTFUM21_6_13 in Fig. 2). In order to identify
which of g.888G>C (p.D261H) or g.2551G>T (p.G678*) (Fig.
3) is the causative mutation of fumonisin non-production in
G1, complementation of FUM21 with either of these point
mutation was conducted in G1.The plasmid that carry a point(pDT21G888C-1)
and
2551thmutation
at
888th(pDT21G2551T-2) nucleotide in FUM21 of G9 was created
and transformed into FfT67831011213-30 (a transformant of
G1 carrying FUM6/ FUM7/ FUM8/ FUM10/ FUM11/ FUM2/
FUM13 regions of G9) (Fig. 2). None of the transformants
recovered fumonisin production in case of pDT21G2551T-2(FfDTFUM21G2551T_6_13 in Fig. 2) while five

MATERIALS AND METHODS¾¾¾

medium. RT-PCR products were subjected to 2%
electrophoresis gel.
FUM21 from G9 was cloned into a transformation
plasmid vector. Point mutation was also created in
FUM21 of the plasmid.
Fumonisin production recovery of transformants wasdetermined by ELISA (detection limit is 0.22 ppm) with
culturing in corn medium for 10 days in 25º C.
Fumonisins in three of each transformant series including
previous studies (Kobayashi 2013) were quantified by
LC-MS/MS analyses.
PCR-RFLP (Restricted Fragment Length Polymorphism)
was developed to determine g.2551 G> T (p.G 678 *) of
FUM21 of F. fujikuroi using dCAPS primer.

Ninety five Fusarium fujikuroi strains, fumonisin
production of that had been investigated in previous
report were used (Suga et al. 2014).
Genomic DNA was extracted from 3–4-day old
mycelium (2–3 cm diameter) cultured on potato dextrose
broth (PDB) by using potassium ethyl xanthogenate
solution, as previously described (Suga et al. 2014).
Confirmed transcription of some FUM gene of G1 andG9 by RT-PCR after culturing by fumonisin induction108Proceedings of International Symposium on Animal Production and Conservation for Sustainable Development 2018
UGSAS-GU & BWEL Joint Poster Session on Agricultural and Basin Water Environmental Sciences 2018: P14

transformants recovered fumonisin production in case ofpDT21G888C-1 (FfDTFUM21G88C_6_13 Fig. 2). These
results suggested that g.2551G>T (p.G678*) rather thang888G>C (p.D261H) is one of the cause of fumonisin nonproduction in G1 (Fig. 3).

Fig. 2: Result of FUM gene complementation in G1 bytransformation (the number of fumonisin producing
transformants/ the number of investigated transformants).
Here, “T” is DNA fragment from fumonisin producing
strain G9.

According to our previous study multiple causative mutations
are present in G1 and g.2551G>T (p.G678*) is one of them. In
future we further identify additional mutations and thevariation of mutations in G-group strains.

REFERENCES
1. Bolton, S. L., Brannen, P. M. and Glenn, A. E. (2016). A
novel population of Fusarium fujikuroi isolated fromsoutheastern U.S. winegrapes reveals the need to re-evaluate
the species’ fumonisin production. Toxins 8: 1-14.
2. Brown, D. W., Butchko, R. A., Busman, M. and Proctor, R.
H. (2007). The Fusarium verticillioides fum gene cluster
encodes a Zn(II)2Cys6 protein that affects FUM gene
expression and fumonisin production. Eukaryotic Cell 6:1210-1218.
3. Cruz, A., Marín, P., González-Jaén, M. T., Aguilar, K. G. I.
and Cumagun, C. J. R. (2013). Phylogenetic analysis,
fumonisin production and pathogenicity of Fusarium fujikuroi
strains isolated from rice in the Philippines. Journal of Scienceof Food and Agriculture 93: 3032–3039.
4. Marasas, W. F. O., Riley, R. T., Hendricks, K. A., Stevens,
V. L., Sadler, T. W., Gelineau-van, Waes J., Missmer, S. A.,
Cabrera, J., Torres, O., Gelderblom, W. C. A., Allegood, J.,
Martinez, C., Maddox, J., Miller, J. D., Starr, L., Sullards, M.
C., Roman, A. V., Voss, K. A., Wang, E. and Merrill, A. H.,
Jr. (2004). Fumonisins disrupt sphingolipid metabolism, folatetransport, and neural tube development in embryo culture andin vivo: A potential risk factor for human neural tube defects
among populations consuming fumonisin-contaminated maize.
Journal of Nutrition 134:711-716.
5. Proctor, R. H., Brown, D. W., Plattner, R. D. andDesjardins, A. E. (2003). Co-expression of 15 contiguous
genes delineates a fumonisin biosynthetic gene cluster inGibberella moniliformis. Fungal Genetics and Biology 38:
237-249.
6. Rheeder, J. P., Marasas, W. F. O. and Vismer, H. F. (2002).
Production of fumonisin analogs by Fusarium species.
Applied and Environmental Microbiology 68: 2101-2105.
7. Rosler, S. M., Sieber, M. K. C., Humpf, H-U. andTudzynki, B. (2016). Interplay between pathway-specific andglobal regulation of the fumonisin gene cluster in the ricepathogen Fusarium fujikuroi. Applied Microbiology andBiotechnology 100: 5869-5882.
8. Suga, H.; Kitajima, M.; Nagumo, R.; Tsukiboshi, T.;
Uegaki, R.; Nakajima, T.; Kushiro, M.; Nakagawa, H.;
Shimizu, M. and Kageyama, K. (2014) A single nucleotide
SRO\PRUSKLVP LQ WKH WUDQVODWLRQ HORQJDWLRQ IDFWRU Į JHQH
correlates with the ability to produce fumonisin in Japanese
Fusarium fujikuroi. Fungal Biology 118: 402–412.

Fig. 3: Comparison of the amino acid sequences of FUM21
of fumonisin producing strain (upper) G9 and fumonisin
non-producing G1 (lower).
Frequency of FUM21 mutation in F. fujikuroi strains
We developed PCR-RFLP that can determine g.2551G>T in
FUM21 (Fig. 4).

Fig. 4: PCR-RFLP for determination ofg.2551G>T in FUM21.
All F-group strains tested showed uncut type (i.e. G type)whereas 35 (81%) out of 44 G-group strains showed cut type(T type) (Table 1).
Table 1. Frequency of FUM21 mutation in F. fujikuroi
strains.

Table 1 (continue)

109

Proceedings of International Symposium on Animal Production and Conservation for Sustainable Development 2018
UGSAS-GU & BWEL Joint Poster Session on Agricultural and Basin Water Environmental Sciences 2018: P15

Suitable culvert configuration for prevention the risk of dam failure due tohydraulic fracturing adjacent to culverts in embankment dams
Duy Quan Tran1, Shinichi Nishimura2, Masateru Semge2 and Tatsuro Nishiyama2
1. The United Graduate School of Agricultural Science, Gifu University, Japan.
2. Faculty of Applied Biological Sciences, Gifu university, Japan.
modified by a combination of inclining chamfers (IG=1.0) andslanted walls with a gradient (G).

INTRODUCTION
Hydraulic fracturing is generally considered as one of themost probable causes leading to the concentrated leakage andincidents of many fill dams soon after the completion of the
dam’s construction (Sherard et el. 1972). For predicting theoccurrence of hydraulic fracturing at any point in a fill dam,the normal stress of the point is compared with the waterpressure at that point. If the water pressure is higher than thenormal stress, it is thought that hydraulic fracturing has
occurred. Previous studies revealed that the risk of hydraulicfracturing increases in cases where the arching action ispresent (Ngambi et al. 1997). Due to this arching action, thestress in fill dams can be reduced to a level lower than thewater pressure.
It is generally identified that there are high risks of dam
incidences resulted from arching action and hydraulicfracturing adjacent to the culverts of the fill dams whereelastic moduli between the culvert materials and the fill soils
are significantly different (Sherard et el. 1972, Ngambi et al.
1997). Hence, selection a suitable culvert configuration for
reducing the risk of hydraulic fracturing is truly necessary.
Culverts utilized in practice generally have a pipe shape, a box
shape or a horseshoe shape. Past researches have revealed,
however, that using culverts with these shapes brings about
the potential risk for inducing arching action and hydraulicfracturing (Ngambi et al. 1997, Tran et al. 2018). The purposeof this study is to find the relationship between culvert
configurations and arching action as well as the risk ofhydraulic fracturing adjacent to culverts by using finite
element analyses. The results of the simulations suggest somesuitable culvert shapes to prevent hydraulic fracturing aroundthe culverts of agricultural dams with low to medium heights.

Table 1: Material properties
Total density (ȡ):
Dry density (ȡd):
Coefficient of permeability (k):Elastic modulus (E):
Poisson’s ratio (Ȟ):

2.018 Mg/m3
1.673 Mg/m3
6.247×10-5 cm/s
16800 kPa
0.3

Inclining chamfer (IG=1.0)

1.1m

1.1m

Dimension 0.1m x 0.1m

1.2m

1.2m

a)

0.4m

b)

1.2m

0.4m

Inclining chamfer (IG=1.0)
Dimension 0.1m x 0.1m

1.1m

Slanted wall
Gradient (G)MATERIALS AND METHODSc)

Materials—To investigate the effects of the different
culvert shapes on the risk of hydraulic fracturing in fill dams,some background information on a dam failure was utilized
here as a case study. The case study dam is an agricultural
dam called KE 2/20 REC dam in Central Vietnam. The dam
was put into operation in July 2008. However, in June 2009 –just under one year after it was put into operation - the dam
broke at the location of the pipe culvert. Previous study hassuggested that the cause of the dam failure is associated withthe hydraulic fracturing and arching action occurring adjacentto the pipe culvert. In this study, therefore, thephysicomechanical properties of the fill soil material is takenfrom the previous investigation and are summarized in Table 1(Tran et al. 2018).
Figure 1 shows the culvert shapes considered in this study.
The dimensions of these culverts, including the height of 1.1
m and the width of 1.2 m, are kept to be similar to those of thepipe culverts in the case study dam. In Fig. 1a, a box-shaped
culvert is displayed. In the case of Fig. 1b, the box-shaped
culvert (Fig. 1a) is changed by inclining the chamfers(IG=1.0) in variations of 0.1 m in the vertical direction and 0.1
m in the horizontal direction. In Fig. 1c, the culvert shape is

Fig.1: Cross section of culvert
Numerical analysis—The finite element method (FEM) is
often utilized in studies of the stress-strain distribution in dams
as well as in investigations of hydraulic fracturing in fill dams.
Based on finite element analyses, the stress distribution arounda culvert can be determined, after which the possibility of
hydraulic fracturing can also be predicted by comparing thestress with the water pressure at corresponding locations. In
this research, therefore, build-up analyses with 6 layers of fillsoil using FEM was simulated to evaluate the likelihood of
hydraulic fracturing around culverts with different
configurations. Figure 2 shows the finite element mesh for arepresentative case of the culvert shape (Fig. 1c) containing
the inclining chamfers (IG=1.0) and the slanted walls with agradient of G=0.4. This model consists of 1024 elements and3201 nodal points. All elements are eight-node quadratic
quadrilateral elements and elements adjacent to culvert havesmaller dimension than the others in order to improveaccuracy and detail of stress distribution around the culvert.

110Proceedings of International Symposium on Animal Production and Conservation for Sustainable Development 2018
UGSAS-GU & BWEL Joint Poster Session on Agricultural and Basin Water Environmental Sciences 2018: P15

9 A

Y

minimum value for (ın-W) increases when the gradient of theslanted walls is changed from 0.1 to 0.5. It seems to suggestthat the effect of arching action might be lowered in cases ofhigher gradients of the slanted walls. The results in Fig. 4
also reveal that when the gradients are equal to or higher than0.4, the risk of hydraulic fracturing along the outlet conduits
can be controlled.
400.0

F
6th
5th

Height (m)

4th
3rd

G =0.1

2nd

300.0
0

G =0.2

1st

C
D

0

Width (m)G =0.3
ın-W (kN/m2)

B

E X
10

Boundary conditions:+ AB and EF: X-direction fixed, Ydirection free.
+ BCD: X- and Y- directions fixed.
+ DE: X-direction free and Y-direction
fixed.

G =0.4200.0

G =0.5100.0

0.0

Fig.2: Cross section of culvert-100.0RESULTS AND DISCUSSION

0.0

0.8
1.2
1.6
L (m)
Fig.4: Distribution of normal stress minus water pressure(ın-W) around culverts with inclining chamfers (IG=1.0)
and slanted walls (Fig. 1c)

Potential for hydraulic fracturing adjacent to box-shaped
culverts—The distribution of normal stress minus waterpressure (ın-W) versus the distance along the culvert periphery(L) for the box-shaped culverts (in Figs. 1a and 1b) are shownin Fig. 3. In this figure, the distance (L) is calculated from theorigin at the midpoint of the culvert top (similar to B in Fig.
2). As seen in Fig. 3, in both cases of the box-shaped culverts,
the normal stress on the sides of the culverts are significantly
reduced to be really lower than the water pressure due to theeffect of the arching action. The maximum value of (ın-W) at
the sides of the culverts is about -6 kN/m2. Therefore, it can beconcluded that there are actual risks of hydraulic fracturing
adjacent to the box-shaped culvert as well as the box-shaped
culvert with the inclining chamfers.
500.0

Based on the results discussed in the previous section, thefollowing conclusions are drawn:(1) There is a potential for hydraulic fracturing close to boxshaped culverts and box-shaped culverts with inclining
chamfers because the normal stress on the sides of the culverts
is reduced by arching action to point of being lower than thewater pressure.
(2) Culverts with slanted walls can reduce the risk ofhydraulic fracturing compared with culverts with verticalwalls. In the Japanese standard, the slanted walls withgradient’s values from 0.1 to 0.3 are suggested for applying toconstruction. Based on the simulated results in previous
sections, however, we recommend that the gradient of 0.4 orhigher values should be applied to practice to control thepotential for hydraulic fracturing around the culverts.

IG =0.0

300.0

ın-W (kN/m2)

CONCLUSION

IG =1.0

400.0

0.4

200.0
100.0

REFERENCES

0.0

Ngambi S., Nakano R., Shimizu H. and Nishimura S. (1997)
Cause of leakage along the outlet conduit underneath a low
fill dam with special reference to hydraulic fracturing,
JSIDRE, No.188, pp.93-102.
Sherard J. L., Decker R. S., and Et-ryker N. L. (1972)
Hydraulic fracturing in low dams of dispersive clay,
Proceedings of ASCE Specialty Conference on theperformance of earth and earth supported structures,pp.653-689.
Tran D. Q., Nishimura S., Senge M., and Nishiyama T. (2018)
Research on the cause of dam failure from viewpoint ofhydraulic fracturing – a Case study of a dam failure in
Vietnam, International Journal of GEOMATE, Vol. 14,
Issue 41, pp.86-94.

-100.0-200.0
0.0

0.4

0.8
1.2
1.6
L (m)
Fig.3: Distribution of normal stress (ın) minus waterpressure (W) around box-shaped culvert (Fig. 1a – IG=0.0)
and box-shaped culvert with inclining chamfers (Fig. 1b –IG=1.0)Effect of the gradient of slanted walls on the risk ofhydraulic fracturing—Figure 4 shows the distribution of (ınW) around the culvert shape in Fig. 1c, including theinclining chamfers (IG=1.0), and the slanted walls withgradients varying from 0.1 to 0.5. It can be seen that the111

Proceedings of International Symposium on Animal Production and Conservation for Sustainable Development 2018
UGSAS-GU & BWEL Joint Poster Session on Agricultural and Basin Water Environmental Sciences 2018: P16

Expression GWAS-An approach for understanding the ALS3 Signal
Transduction Mechanism
Raj Kishan Agrahari1*, Ayan Sadhukhan2*, Yuki Nakano1, Satoshi Iuchi3, Masatomo Kobayashi3, Sanjib Kumar
Panda4, Yuriko Kobayashi1, 2 and Hiroyuki Koyama1, 2
1.
The United Graduate School of Agricultural Science, Gifu University
2.
Faculty of Applied Biological Sciences, Gifu University, Gifu 501- 1193, Japan
3.
Expermental Plant Division, RIKEN Bio Resource Centre, Tsukuba, Ibaraki 305-0074, Japan
Department of Life Science, Assam University, Silchar 788011, India
4.
*These authors contributed equally to this work

INTRODUCTION

Plant Growth Condition

Aluminum (Al) toxicity in acid soils (pH < 5.5) is a severe
global problem that affects greater than 30% of the world’s
arable land, with this being a major limiting factor for crop
productivity in these areas (von Uexkull and Mutert, 1995).
Physiologically Al toxicity includes severe root growth
inhibition coupled with reduction in shoot biomass and crop
yield (Kochian, 1995). At the cellular level, Al toxicity resultsfrom deleterious effects of the Al3+ ion, which is the toxic
species of Al that predominates under acidic conditions. Al3+
targets are both external, including the cell wall (Tabuchi andMatsumoto, 2001), and intracellular including thecytoskeleton (Blancaflor et al., 1998; Grabski and Schindler,
1995), ion transporters (Huang et al., 1992), constituents ofsignaling systems (Haug et al., 1994; Jones and Kochian,1995), and various other cellular components and enzymes(Jones and Kochian, 1997), many of which are negatively
charged or coordinate positively charged cofactors (Kochian,1995).

Germinated A.thaliana seedlings were grown on mesh andtransferred to to modified 2% MGRL (pH was 5.6) culture
solution (Fujiwara et al., 1992). The solution was changed
every 2 days, and grown under controlled environmental
conditions for 10 days (Sawaki et al., 2016).
Gene Expression Analysis
10 days old seedlings were then transferred to the treatment
VROXWLRQ FRQWDLQLQJ  ȝ0 $O DIWHU D -h stress treatment,
the shoots were collected and freeze in liquid nitrogen, andcrushed using tissue homogenizer. Total RNA was extracted
from the shoot and used for cDNA. ALS3 expression level was
determined by real-time qPCR.
Genome-wide Association Study
The GWAS calculations were completed using thecompressed mixed linear model (Yu et al., 2006; Zhang et al.,2010) of the TASSEL program as previously described(Kobayashi et al., 2016). The genome-wide SNPs used in thisstudy were obtained from a public database. Missing data or aminor allele frequency below 10% was removed. Genes
nearest to the most significantly associated SNPs wereannotated according the TAIR 10 database.

Previously, a molecular genetics approach was
employed using Arabidopsis thaliana as a model system inorder to identify factors required for Al resistance and/or
tolerance in plants (Larsen et al., 1996, 1997). From thisapproach a unique als-3 mutant was found to comprise with
distinct characteristics (Larsenet al., 1996). Al-dependent
phenotypes of als3 include complete arrest growth of the
primary root, lateral roots, and the shoot. ALUMINIUMSENSITIVE 3 (ALS3) encodes a half type ATPase is involvedin the Al-translocation process, while the gene is inducible inthe shoots under Al stress, this gene is involved in theredistribution of toxic Al away from sensitive tissues thereby
conferring Al tolerance. But the detailed regulatory
mechanisms leading to the induction of ALS3 under Al stressremain unclear. Here we present our work regarding the newsignaling molecules upstream to ALS3.

ALS3 expression analysis in KO line
Total RNA was extracted from a 24-K VWUHVV  ȝ0 $O 
treated shoot sample, and used for cDNA. ALS3 expression
level was determined by real-time qPCR.

RESULTS & DISCUSSION
From a GWAS on ALS3 expression we found 22 SNPs
(P<103) to be strongly associated to ALS3 expression. Role of
the genes, linked to the most significant SNPs, in regulating
ALS3 was validated by reverse genetics.

MATERIALS AND METHODS
Plant Materials

Number of A.thaliana
accessions

Seeds of natural A.thaliana accessions and KO lines wereobtained from the Arabidopsis Biological Resource Center(Columbus, OH, USA), the Nottingham Arabidopsis Stock
Center (Nottingham, UK), and the RIKEN BioResource
Center (Tsukuba, Japan), We treated 100 A.thaliana
accessions wiWKȝ0$OIRUWKH*:$63URJHQ\OLQHVZHUH
obtained by controlled self-pollination method. The
homozygocity of KO lines was confirmed by PCR using
SALK recommended methods.

112

20
15
10
5
0

A

0

0.2 0.4 0.6 0.81

1.2 1.4

Relative Fold Change of ALS3

ALS3/UBQ1

Proceedings of International Symposium on Animal Production and Conservation for Sustainable Development 2018
UGSAS-GU & BWEL Joint Poster Session on Agricultural and Basin Water Environmental Sciences 2018: P16

1.2
1
0.8
0.6
0.4
0.2
0

Tabuchi, A. and Matsumoto, H. (2001) Changes in cellwall properties of wheat (Triticum aestivum) roots
during aluminum- induced growth inhibition. Physiol.
Plant. 112,
353–358.
Von Uexkull, H.R. and Mutert, E. (1995) Global extent,
development and economic impact of acid soils. Plant
Soil, 171,1–15.

B

Fig 1. (A)Histogram of ALS3 expression in A. thaliana
accessions. (B) Expression of ALS3 in T-DNA insertion
mutants of GWAS detected genes.
As ALS3 encodes a putative ABC transporter homologous toa predicted bacterial metal resistance protein, responsible for Al
tolerance, though it’s signaling is unclear. We did GWAS to
identify a signaling cascade regulating ALS3 expression under
Al stress. From the GWAS we successfully identified ALS3
signaling cascade that provide genetic evidence of theinvolvement of phospholipids signaling under Al stress.

REFERENCES
Blancaflor, E.B., Jones, D.L. and Gilroy, S. (1998) Alterations in
the cytoskeleton accompany aluminum-induced growth
inhibition and morphological changes in primary roots ofmaize. Plant Physiol. 118, 159–172.
Fujiwara, T., Hirai, M.Y., Chino, M., Komeda, Y., and Naito, S.
(1992). Effects of sulfur nutrition on expression of the soybean
seed storage protein genes in transgenic petunia. Plant Physiol.
99:263–268.
Grabski, S. and Schindler, M. (1995) Aluminum induces rigor
within the actin network of soybean cells. Plant Physiol. 108,
897–901.
Haug, A., Shi, B. and Vitorello, V. (1994) Aluminum interaction
with phosphoinositide-associated signal transduction. Arch.
Toxicol. 68, 1–7.
Huang, J.W., Grunes, D.L. and Kochian, L.V. (1992) Aluminum
effects on the kinetics of calcium uptake into cells of the wheatroot apex. Quantification of calcium fluxes using a calcium
selective vibrating microelectrode. Planta, 188, 414–421.
Jones, D.L. and Kochian, L.V. (1997) Aluminum interaction withplasma membrane lipids and enzyme metal binding sites anditspotential role in Al cytotoxicity. FEBS Lett. 400, 51–57.
Kochian, L.V. (1995) Cellular mechanisms of aluminum toxicity
and resistance in plants. Annu. Rev. Plant Physiol. Plant Mol.
Biol. 46, 237–260.
Larsen, P.B., Tai, C.Y., Kochian, L.V. and Howell, S.H. (1996)
Arabidopsis mutants with increased sensitivity to aluminum.
Plant Physiol. 110, 743–751.
Larsen, P.B., Kochian, L.V. and Howell, S.H. (1997) Al inhibits
both shoot development and root growth in als3, an Al-sensitive
Arabidopsis mutant. Plant Physiol. 114, 1207–1214.
Sawaki,K.,Sawaki.Y.,Zhao,C.R.,Kobayashi,Y.,andKoyama,H.(2016) Specific transcriptomic response in the shootsofArabidopsis thaliana after exposure to Al
rhizotoxicity: potential gene expression biomarkers for evaluating Al toxicity
in soils. Plant Soil. 409:131–142

113

Proceedings of International Symposium on Animal Production and Conservation for Sustainable Development 2018
UGSAS-GU & BWEL Joint Poster Session on Agricultural and Basin Water Environmental Sciences 2018: P17

A Survey of GABA production from different species of Bifidobacterium
Hend Al taib 1, Takumi Morioka 2, Mayuko Abe 3, Izumi Nomura 1 and Tohru Suzuki1,2
1. The United Graduate School of Agricultural Science
3. Faculty of Applied Biological Sciences
2. Graduate School of Natural Science and Technology
HPLC condition has been managed as follow; Reverse
phase chromatography with cadenza column CD-C18
INTRODUCTION(250x4.6mm i.d.,3m), Column oven at 35°C, flow rate
0.8ml/min. A gradient system elute has been used (waterBifidobacterium is one of the most important probiotics
acetonitrile, 2-propanol, TFA) The NBD-F pre-column
which normally inhabits the gut of all mammals including
derivatization has been used for GABA production
animal and human. It exerts its function mainly throughestimation according to the method by (C Aoyama et al.
existence as a part of the gut microbiota. Gut microbiota2003). Each sample has been prepared in triplicate.
represents 10 times as many cells as that in our body and 150
Biological error has also been considered by the triplicate
times as that of our genomes. Therefore, it is often called theculture of each sample.
forgotten organ.
4. In silico analysis of Gad gene prevalence andRecent years have witnessed the rise in the gut microbiota
homology in bifidobacterium producer strains.
brain axis as a major topic of interest in biology (Grenham etUsing CLC working bench ver.8, the multigene
al 2011). Gut-brain axis refers to the interplay between gut andalignment has been performed for gad genes. NCBI has beenbrain which occurs through many pathways. One connectionused to obtain the available genome sequences. Wholepathway occurs through neurotransmitters which transfer fromgenome sequence analysis has been performed using Illumina
gut to brain and vice virsa by specific signals. These signals
Miseq analyzer of GIFU University for one high producer andpass between the enteric nervous system of the gut and theanother nonproducer strain of bifidobacteria.
central nervous system (CNS) of the brain. Recently microbes
have proved to produce several neurochemical transmitterswhich drive the name microbial endocrinology (Lyte et al
RESULTS and DISCUSSION
2011). Those materials have the same structure as that of
1. B. adolescentis shows the highest GABA production.
mammals. Such producing microbes are also named
psychobiotics (Dinan et al 2013).
GABA standard curve has been estimated with
Bifidobacterium has proved to produce several
equation y=443.34x, R2=0.998. The values werenormalized considering the internal standard. B.
neurotransmitters.
One
of
the
most
importantneurotransmitters, recently shown to be produced byadolescentis laboratory strain no.4-2 has shown to bebifidobacteria, is Gamma-aminobutyric acid (GABA). GABA
the highest in GABA production up to about 3 g/L
is the major inhibitory neurotransmitter in the CNS. It also has(after two overnight incubation). The whole genome
an immunomodulatory function through the existence of its
sequence of this strain has been done and gadB andreceptors in most of the immune cells (Jin et al 2013).
gadC have been found in the genome. Another strain
Basically, GABA is biosynthesized by decarboxylation of
B. adolescentis JCM 1275 has shown zero production
glutamate by the action of glutamate decarboxylase encodedof GABA and the whole genome data also shows theby gadB and transferred to outside of the cell throughabsence of gadB encoding Glutamate decarboxylase
glutamate/Gamma amino butyrate antiporter encoded by
and gadC encoding glutamate/GABA antiporter genes
gadC(R Dhakal et al 2012). As it has an important function onwhich means those two genes are essential for GABA
both neuro-immunomodulation level and little investigations
production in genus Bifidobacterium. This data
are available about its productivity from Bifidobacterium, aconfirms the result obtained by the Russian researchdetailed study for its production from bifidobacterium genes isgroup (R.A. Yunes et al 2016)needed. Here, A survey of GABA production ability and2. Manno Oligo Saccharides (MOS) have shown to
producing gene prevalence within the genus Bifidobacterium
enhance GABA productivity in flora model.
has been performed.
The MOS has enhanced GABA production comparedto Faracto Oligo Saccharides (FOS)and Dextrins (Dex)table1. This could be referring to the ability of
MATERIALS AND METHODS
bifidobacteria to digest Oligosaccharides into shortchain fatty acids.
1. Bacterial strains and culture condition.
More than 25 different bifidobacteria species have been
The added oligosaccharide Normalized GABA peak
selected from the National Bio Recourse Project (NBRP),
g/L
strains have been selected according to the homology ofControl (no additives)
99.2
gad genes. MRS was supplemented with 1% monosodium
glutamate. Monosodium glutamate acts as a precursor of
Flora model+0.5%MOS
145
GABA in the media.
Flora model+0.5%Dex
82.1
2. Selection a suitable prebiotic enhancer by using flora
Flora model+0.5%FOS
146model to mimic in vivo environment.
To maintain high GABA production on a long run in the
Table.1. the estimated GABA production in flora
gut, it was needed to select a good prebiotic enhancer. Floramodel under addition of various resources ofmodel (Takagi et al. 2016) has been selected to mimicoligosaccharides, the process estimated for fecal
inside environment for a selection of best prebiotic
samples from bifidobacteria carrier volunteers. It
enhancer. Different resource of oligosaccharides has been
confirms that MOS and FOS are two good prebiotic
added to flora model jars. The final GABA production has
enhancer for bacterial growth in flora model which
been estimated with HPLC
mimic the gut environment.
3. HPLC Screening of GABA production.

114

Proceedings of International Symposium on Animal Production and Conservation for Sustainable Development 2018
UGSAS-GU & BWEL Joint Poster Session on Agricultural and Basin Water Environmental Sciences 2018: P17

3.

A high homology gene set for GABA production exists
within the genus Bifidobacterium.
The multigene alignment shows high homology in thesequences of gadB genes which confirm that this geneis essential for GABA production in the genus
Bifidobacterium. The pyridoxal phosphate binding
motif PLP binding motif has also confirmed to behomologues in this gene. PLP is an important cofactor
for glutamate decarboxylase activity. Once it is foundmeans this gene should have some activity. The
multigene alignment data is shown in fig.1.

Dhakal, R., Bajpai, V. K., & Baek, K. H. (2012). Productionof GABA (Ȗ-aminobutyric acid) by microorganisms: areview. Brazilian Journal of Microbiology, 43(4), 12301241.
Takagi, R., Sasaki, K., Sasaki, D., Fukuda, I., Tanaka, K.,
Yoshida, K. I., ... & Osawa, R. (2016). A single-batch
fermentation system to simulate human colonic microbiota
for high-throughput evaluation of prebiotics. PLoS
One, 11(8), e0160533.
Aoyama, C., Santa, T., Tsunoda, M., Fukushima, T., Kitada,C., & Imai, K. (2004). A fully automated amino acidanalyzer using NBDǦF as a Àuorescent derivatization
reagent. Biomedical Chromatography, 18(9), 630-636.
Yunes, R. A., Poluektova, E. U., Dyachkova, M. S., Klimina,
K. M., Kovtun, A. S., Averina, O. V., ... & Danilenko, V. N.
(2016). GABA production and structure of gadB/gadC
genes in Lactobacillus and Bifidobacterium strains fromhuman microbiota. Anaerobe, 42, 197-204.

REFERENCES
Grenham, S., Clarke, G., Cryan, J. F., & Dinan, T. G. (2011).
Brain̽gut–microbe communication in health anddisease. Frontiers in Physiology, 2, 94.
Lyte, M. (2011). Probiotics function mechanistically as
delivery vehicles for neuroactive compounds: microbial
endocrinology in the design and use of probiotics.
Bioessays, 33(8), 574-581.
Dinan, T. G., Stanton, C., & Cryan, J. F. (2013).
Psychobiotics: a novel class of psychotropic. BiologicalPsychiatry, 74(10), 720-726.
Jin, Z., Mendu, S. K., & Birnir, B. (2013). GABA is aneffective immunomodulatory molecule. Amino Acids, 45(1),87-94.

Fig.1 Multifragment alignment for gadB gene in 10 strains of bifidobacteria. It shows a high consensus sequence of gadB gene andalso a high similarity in PLP binding motif marked by the gray rectangular in the upper part of the figure. The software used was
CLC working bench ver.8.

115Proceedings of International Symposium on Animal Production and Conservation for Sustainable Development 2018
UGSAS-GU & BWEL Joint Poster Session on Agricultural and Basin Water Environmental Sciences 2018: P18

Identification of bacteria from bioaerosol at AIST, Tsukuba, Japan
Panyapon Pumkaeo, Wenhao Lu, Youki Endou, Tomohiro Mizuno, Junko Takahashi and Hitoshi Iwahashi
The United Graduate School of Agricultural Science, Gifu University
Faculty of Applied Biological Sciences, Gifu University

INTRODUCTION
Bioaerosols are atmosphere particles, mists or dust of
m range, associated with metabolically active orinactive viable particles. They contain living organism’s
included microorganisms such as viruses, bacteria, andfungi also plant material as well as pollen. For study
about microbial diversity in human and environment,
Next Generation Sequencing (NGS) become a famous.
NGS is a novel method of DNA sequencing that quickly
and efficiently read the underlying sequence of anorganism by means of massively parallel sequencing.
The aim of this study is identifying bacteria whichcontained in environmental samples by using NGS.

100%
90%
80%

Relative abundance (%)

70%
60%
50%
40%
30%
20%
10%

MATERIALS AND METHODS

Sample2_MID-107

Sample2_MID-106

Sample2_MID-105

Sample1_MID-107

Fig. 1:

Sample1_MID-106

This study monitored the environmental sample(Bioaerosols) from November 2013 to January 2014 for
50 days using air samples were collected at AIST,
Tsukuba, Japan. Samples were bio-analyzed using anext-generation sequencing method. In this study, weused two NGS platform. The sample was detectedbacteria. The sample was divided into three subgroupand two subgroups total 6 samples according to the sizeof its bioaerosols, large subgroup contains bioaerosols
ZKRVH GLDPHWHU LV ELJJHU WKDQ ȝP DQG VPDOOVXEJURXSFRQWDLQVWKRVHVPDOOHUWKDQȝPSample1_MID-105

0%abundant bacterial groups
identified
using
Next
Generation Sequencing at thelevel.

no_rank
Verrucomicrobiae
Thermotogae (class)
Thermomicrobia (class)Spirochaetia
Sphingobacteriia
Spartobacteria
Solibacteres
Planctomycetacia
Opitutae
Nitrospira (class)
Negativicutes
Mollicutes
Ktedonobacteria
Holophagae
Gemmatimonadetes (class)Gammaproteobacteria
Fusobacteria (class)
Flavobacteriia
Fibrobacteres (class)
Erysipelotrichi
Epsilonproteobacteria
Deltaproteobacteria
Deinococci
Dehalococcoidetes
Deferribacteres (class)
Cytophagia
Clostridia
Chthonomonadetes
Chloroflexi (class)
Betaproteobacteria
Bacteroidia
BacilliArmatimonadia
Anaerolineae
Alphaproteobacteria
Actinobacteria (class)
Acidobacteria (class)

Most

class

Fig. 2: Most abundant bacterial groups identified using Next
Generation Sequencing at the family level.
100%

RESULTS

90%

There are three groups of bacteria samples.
Each collected in different time. Hits by days shows
found that quantities of bacteria DNA appear equally inthe bioaerosol samples. Results of taxonomy of bacteria,
the 16s rDNA sequences were divided into 505
phylotype. (data not show) The bacteria composition
was included in 41 class. (Figure.1) The main classwere Actinobacteria, Alphaproteobacteria, Bacili andClostridia that found in the all sample.181 Family wereclassified. (Figure.2) The high abundance of bacteria
wasClostridiace,
Lactobacilliacae
andMethylococaceae. We can see that results of bacteria
identification have no distinctions neither in different
period nor between smaller and larger subgroups thatmeans bacteria are not suitable for being regionalspecific and seasonal-specific organisms.

80%

Relative abundance (%)

70%

60%

50%

40%

30%

20%

10%

116

Sample2_MID-107

Sample2_MID-106

Sample2_MID-105

Sample1_MID-107

Sample1_MID-106

Sample1_MID-105

0%

no_rank
Yaniellaceae
Xanthomonadaceae
Xanthobacteraceae
Williamsiaceae
Victivallaceae
Vibrionaceae
Verrucomicrobiaceae
Verrucomicrobia subdivision 3
Veillonellaceae
Trueperaceae
Thermotogaceae
Thermomonosporaceae
Thermoanaerobacteraceae
Thermoactinomycetaceae
Syntrophomonadaceae
Succinivibrionaceae
Streptosporangiaceae
Streptomycetaceae
Streptococcaceae
Staphylococcaceae
Sporolactobacillaceae
Spiroplasmataceae
Spirochaetaceae
Sphingomonadaceae
Sphingobacteriaceae
Sphaerobacteraceae
Solirubrobacteraceae
Solibacteraceae
Sinobacteraceae
Shewanellaceae
Scytonemataceae
Saprospiraceae
Sanguibacteraceae
Sandaracinaceae
Ruminococcaceae
Rubrobacteraceae
Rivulariaceae
Rikenellaceae
Rickettsiaceae
Rhodothermaceae
Rhodospirillaceae
Rhodocyclaceae
Rhodobacteraceae
Rhizobiaceae
Puniceicoccaceae
Pseudonocardiaceae
Pseudomonadaceae
Propionibacteriaceae
Promicromonosporaceae
Prevotellaceae
Porphyromonadaceae
Polyangiaceae

Proceedings of International Symposium on Animal Production and Conservation for Sustainable Development 2018
UGSAS-GU & BWEL Joint Poster Session on Agricultural and Basin Water Environmental Sciences 2018: P18

DISCUSSION

REFERENCE
Maki, T., Hara, K., Iwata, A., Lee, K. C., Kawai, K.,
Kai, K., . . . Iwasaka, Y. (2017). Variations inairborne bacterial communities at highaltitudes over the Noto Peninsula (Japan) in
response to Asian dust events. Atmospheric
Chemistry Physics, 11877-11897.
Núñez, A., Paz, G. A., Ferencova, Z., Rastrojo, A.,
Guantesd, R., García, A. M., . . . Moreno, D.
A. (2017). Validation of the Hirst-type spore
trap for simultaneous monitoring ofprokaryotic and eukaryotic biodiversity inurban air samples by NGS . Appl. Environ.
Microbiol. .
Srivastava, A., Singh, M., & Jain, V. K. (2012).
Identification and characterization of sizesegregated bioaerosols at Jawaharlal Nehru
University, New Delhi . Nat Hazards , 485499.

For bacteria, the hits of read by days was showed thatquantities of bacteria DNA appear equally except group 2 inwhich smaller subgroup contains only half amounts of DNA
comparing to larger subgroup (data not show) Althoughseveral factors may interfere the sampling and DNA
extraction. (Núñez, et al., 2017) found that depletion in the
airflow rate in sample during collection caused of different of
DNA concentration in the replicates. In this study, we tend tobelieve that it is mis-operation during DNA extraction thatcaused deviation. In the dust samples collected at AIST,
Actinobacteria, Alphaproteobacteria, Bacili and Clostridiathat found in the all samples. (Figure1) Common in the aerosol
Bacilli were identified. (Srivastava, Singh, & Jain, 2012)
Some of Actinobacteria were detected in the dust event
samples that collected at the high altitudes over the Noto
Peninsula, Japan. (Maki, et al., 2017) In this study the
information of bacteria in the aerosols and in the future,
biological trace information will be study such as eukaryote,plant and animal in the different areas.

ACKNOWLEDGMENTS
This study is acknowledged for National Institute ofAdvanced Industrial Science and Technology (AIST),
Tsukuba, Japan.

117

Proceedings of International Symposium on Animal Production and Conservation for Sustainable Development 2018
UGSAS-GU & BWEL Joint Poster Session on Agricultural and Basin Water Environmental Sciences 2018: P19

The role of combination of color and scent for attracting pollinators ingeneralist flowers
Muhammad Arifin1 and Tomoko Okamoto2
1. Graduate School of Natural Science and Technology, Gifu University, Japan
2. Faculty of Applied Biological Sciences, Gifu University, Japan
confirmed the presence of pollen grains on the insect body.
Quantification of pollen loads placement on insect body was
also recorded.

INTRODUCTION
Pollination, defined as a transfer of pollen from anther tostigma, is important event of plant life cycle. Plant uses flower
as advertisement for attracting pollinating animals (Fishman
and Hadany 2015). Generally, pollinators use visual signals,
including flower size, color and shape, and olfactory signals,
referred as floral scent, to navigate for visiting flowers, whileplants can attract effective pollinators and avoid inappropriate
flower visitors using visual and olfactory signals (Chen et al.
2010; Okamoto et al. 2015).
The pollination mode is classified into generalized andspecialized pollinating according to the range of pollinating
animals. Generalized pollination defined as use of several to
many species of the available pollinators, in other hand,
specialized pollination use of one or few species of available
pollinators (Ashworth et al. 2004).
Floral color and scent have been focused by many
pollination ecologist as important traits for pollination success(Fenster et al. 2004; Majestic et al. 2007). Some previous
studies revealed that white colored flowers blooming in
daytime can attract a wide variety of pollinators (McCall et al.
2013; Reverte et al. 2016). Several field studies revealed thatcolor morphs exhibited significant differences in floral scentprofiles (Majestic et al. 2007, Majestic et al. 2010). The
phenotypic integration between scent and colour was directly
linked to plant–pollinator interactions (Kantsa et al. 2017). It
is thought that some scent compounds are biosynthesized insame pathway or regulated by same factors with pigments.
However, relationships between the combination of floral
color-scent and type of flower visitors in unclear.
Here we report the progress of field observation to reveal
the pollinator assemblage and flower color of various
flowering plants.

Floral color measurement
In this study, the terms that we used to describe colors werebased on human visual spectrum (~380 nm to 780 nm). DIC
color guide tools were used to measure the petal color of eachspecies.
Floral scent collection and analysis (plan for next season)
We will collect floral scent of each species by head-space
sampling in situ. Floral scent collection will be performed insunny day during the peak of insect visitation (1000 h and1400 h).
Collected samples will be analyzed using gas
chromatography mass spectrometry (GC-MS) to reveal thefloral scent profiles.

RESULTSA wide array of insect visitor in 25 plant species wererecorded. The visitors vary in total number and types between
species (Table 1). Hymenopteran insect, wasps and bees, werethe most common insect in generalist flowers. Moreover, thenumerous flower visitors tend to prefer white colored flower
both in native and invasive flowers. In contrast, invasive
flower species with purple color such as Baptisia australis andVerbena rigida were visited by very few of flower visitors.
Because we are continuing field observation, the statisticalanalysis will be conducted after finishing to collect data.

DISCUSSION
The data of flower visitors, revealed that there was a clear
difference in total numbers and type of insect visitors on eachflower color. The white colored flower tend to attract wider
types of insect visitors with high number than another color.
This result supports the recent studies (McCall et al. 2013)
according to feeding activities of insects, indicating numerous
insects prefer white floral color over others.
Nevertheless, there was no rule such certain color attract
specific type of pollinators. Influence of other floral traits, orthe possibility of trait combinations, seem to have great effectfor attracting the flower visitor. Our temporary result, only
provided from color trait, thus, further studies in floral scentshould be conducted.
Our data also suggest a unique interaction case in invasive
flower species. Invasive flower such as Baptisia australis andVerbena rigida visited by only few insect. In contrast, anotherinvasive species such as Mentha suaveolens attracted a huge
number of insect visitors including native insects. This might
impact directly and indirectly to the change of communitylevel interactions in the ecosystem. To survive and become
successful invader, the invasive flower might construct novel
interaction with resident native iwhich already present in their
new habitat. The pollination in invasive flowers is carried out
by the native pollinator belonging to alike functional group as

MATERIALS AND METHODS
Study plants and field sites
We focused on the flowering plants with generalized
pollination system. Up to now, we observed 25 species of both
native and invasive flowers. The observation was conductedon spring to autumn 2018 during the flowering season of eachspecies. Field site of this study were at several locations in
Gifu Prefecture, Japan.
Collection and analysis of flower visitor
Insect visitors were observed and collected throughout theperiod of flowering season of each species. Because weather
conditions influence the foraging activities of insect, weobserved and collecting flower visitors only in the daytime on
sunny day (0900 h to 1500 h). The observation periods of eachflower vary from 30 minutes to 120 minutes because someflowers showed very low frequency of insect visitation. Thereis no nocturnal observation in this study.
At the field site, flower visitors were collected using hand
net after confirming insects landed on the flowers.
Identification of the specimens was carried out in laboratory.
In order to distinguish the pollinator and flower visitor, we

118Proceedings of International Symposium on Animal Production and Conservation for Sustainable Development 2018
UGSAS-GU & BWEL Joint Poster Session on Agricultural and Basin Water Environmental Sciences 2018: P19

the pollinating plants in their native range (Stout and Tiedeken2016). However, the mechanism of attractiveness for invasive
species to native pollinators deserve further special study.

Kevan, P., Giurfa, M., and Chittka, L. (1996) Why are Thereso Many and so Few White Flowers?. Trends in Plant
Science 1(8): 280-284.
Majetic, C. B., Raguso, R. A., Tonsor, S. J., and Ashman, T.
(2007) Flower Color–Flower Scent Associations in
Polymorphic
Hesperis
Matronalis(Brassicaceae).
Phytochemistry 68:865–874.
Majetic, C. J., Rausher, M. D., and Raguso, R. A. (2010) The
Pigment-Scent Connection: Do Mutations in Regulatory vs.
Structural Anthocyanin Genes Differentially Alter Floral
Scent Production in Ipomoea Purpurea?. South African
Journal of Botany: 76(4):632–642.
McCall, A. C., Murphy, S. J., Venner, C., and Brown, M.
(2013) Florivores prefer white versus pink petal color
morphs in wild radish, Raphanus sativus. Oecologia,
172(1), 189–195.
Okamoto, T., Okuyama, Y., Goto, R., Tokoro, M., and Kato,
M. (2015) Parallel Chemical Switches Underlying
Pollinator Isolation in Asian Mitella. Journal of
Evolutionary Biology 28(3): 590–600.
Reverté, S., Retana, J., Gómez, J. M., and Bosch, J. (2016)
Pollinators Show Flower Colour Preferences but Flowers
with Similar Colours Do Not Attract Similar Pollinators.
Annals of Botany 118(2):249–257.
Stout, J. C., and Tiedeken, E. J. (2016) Direct Interactions
between Invasive Plants and Native Pollinators: Evidence,
Impacts and Approaches. Functional Ecology 31(1):38–46.

ACKNOWLEDGMENTS
We are grateful to Nobumitsu Kawakubo for introducing
the study sites and providing DIC color guide tools.

REFERENCES
Ashworth, L., Aguilar, R., Galetto, L., and Aizen, M. A.
(2004) Why Do Pollination Generalist and Specialist Plant
Species Show Similar Reproductive Susceptibility to
Habitat Fragmentation?. Journal of Ecology 92(4):717–719.
Chen, Y., Li, H. Q., Li, M., and Chen, X., Y.(2010) Species̺
Specificity and Coevolution of Figs and Their Pollinating
Wasps. Biodiversity Science 18:1-10.
Fenster, C. B., Armbruster, W. S., Wilson, P., Dudash, M. R.,
and Thomson, J. D. (2004) Pollination Syndromes andFloral Specialization. Annual Review of Ecology Evolution
and Systematics 35:375–403.
Fishman, M. A., and Hadany, L. (2015) Floral Advertisement
and the Competition for Pollination Services. Biosystems
132-133:35–42.
Kantsa, A., Raguso, R. A., Dyer, A. G., Sgardelis, S. P.,
Olesen, J. M., and Petanidou, T. (2017) Community-WideIntegration of Floral Colour and Scent in a Mediterranean
Scrubland. Nature Ecology & Evolution 1(10):1502–1510.
Table 1. Insect observed visiting flowers at the study sites

Observation
period(Min)

No. ofinsects
visitorTypes of insect visitors

60
60
60
75
60
60

30
31
17
4
34
13

Wasp, bee, beetle, fly
Wasp, bee, beetle, hoverfly, butterfly, bug
Beetle, hoverfly
Wasp, beeWasp, beetle, butterfly, ant, thrips
Wasp, bee, ant

Native

White
White
White
Deep purple blue
Red purple and white
Vivid red purple
Light greenish
yellow

65

25

Wasp, bee, beetle, fly, thrips

NativeInvasive
Invasive

Dull yellow green
White
Pale red purple

70
60
60

46
11
12

Verbena rigida
Leucanthemum vulgare

Invasive
Invasive
Invasive

Vivid reddish purple
Deep purple
White

90
120
60

24
3
18

Wasp, bee, beetle, fly, ant, thrips
Wasp, bee, beetle, fly, hoverfly
Wasp, ant, thrips, homoptera
Wasp, bee, beetle, fly, hoverfly, butterfly,
ant, thrips
Hoverfly, butterfly
Wasp, beetle, ant, thrips, bug

Erigeron philadelphicus
Circum japonicum

Invasive
NativeReddish white
Dull reddish purple

90
120

40
19

Wasp, bee, beetle, fly, hoverfly, butterfly, bugBee, fly, butterfly

Oxalis articulata

Invasive

Vivid red purple
Very pale yellow
greenLight grayish purple
White

60

18

Wasp, bee, beetle, ant

60
30
60

75
40
96

Wasp, beetle, fly, hoverfly, butterfly, ant
Wasp, fly, hoverfly, butterfly, bug
Wasp, beetle, butterfly, thrips, bug, mosquito

90

47

Wasp, bee, fly, hoverfly, butterfly, moth, bug

60
90
70
60
60

75
36
20
69
21

Wasp, bee, fly, ant, thrips
Wasp, bee, beetle, ant, thrips
Wasp, beetle, ant, thrips
Wasp, bug, ant, thrips
Wasp, bee, beetle, fly

Flower species

OriginFlower Colour

Rhaphiolepis indica var.
umbellata
Ligustrum obtusifolium
Photinia glabra
Baptisia australis
Deutzia crenata f. plena
Lespedeza spp.

NativeNativeNativeInvasive
NativeInvasive

Castanea crenata
Ampelopsis glandulosa
var. heterophylla
Ammi majus
Oenothera speciosa
Astragalus sinicus

Euonymus japonicus
Stenactis annuus
Mentha suaveolens

NativeInvasive
Invasive

Salvia lutescens

NativeTaraxacum officinale
Cayratia japonica
Phytolacca americana
Liriope platyphylla
Enkianthus perulatus

Invasive
NativeInvasive
NativeNativeDull purple blue
Vivid greenish
yellow
Yellow green
Pale yellow green
Pale bluish purple
White

119

Proceedings of International Symposium on Animal Production and Conservation for Sustainable Development 2018
UGSAS-GU & BWEL Joint Poster Session on Agricultural and Basin Water Environmental Sciences 2018: P20

Isolation and characterization oF Lactobacillus spp. from virgin coconut
oil, West Sumatera
Yolani Syaputri1,3, Sumaryati Syukur2, Endang Purwati3, Hitoshi Iwahashi1
1. The United Graduate School of Agricultural Science, Gifu University, Yanagido 1-1, Gifu City, 501-1193, Japan
2. Laboratory of Biotechnology, Departement of Chemistry, Faculty of Math and Natural Sciences, University of Andalas,
Padang 25163, Indonesia
3. Laboratory of Biotechnology, Departement of Animal Nutrition, Faculty of Animal Husbandary, University of Andalas,
Padang 25163, IndonesiaUSA), 1.25 L of each primers 27F and 1492R(concentration 0.05 pmol/L), 9 L nuclease free deionizedwater and 1 L template DNA, running under the following
temperature program: initial denaturation of DNA for 5 min
at 95 °C, 25 cycles of 1 min at 94 °C, 1 min at 56°C, and
1.5 min at 72 °C; and final extension for 7 min at 72 °C. 5 laliquots of the PCR products were analyzed byelectrophoresis using 1% (w/v) agarose gel in Tris Acetic
acid EDTA (TAE 1X) buffer at 100 V for 30 min. The gel
was then placed in Electronic U.V. Transilluminator to detect
the presence a band of 1500 bp. The size of the DNA
fragments was estimated using a FastGene 100 bp DNA
Ladder (Nippon Genetics, Germany). Fast Gene™, Gel/PCR
Extraction kit (Nippon Genetics, Germany) was used for
purification before sending the extracted DNA for
sequencing, according to the manufacturer’s instructions. An
average of 500 bp nucleotides for each sequence from eachside was read and compared with the databases using the
BLAST program (http://blast.ncbi.nlm.nih.gov/Blast.cgi).
Isolates with 98% or higher similarity in sequences wereidentified as the same species.

INTRODUCTION
Virgin Coconut oil is an essential oil in West Sumatra andreported to against bacteria, fungi, viruses, and protozoa(Kamdem, Guerzoni, Baranyi, & Pin, 2008). Virgin Coconut
Oil is extracted from the kernel of matured coconut and has
been widely utilized in food, medicine, and industry (HIbrahim, et al., 2017). The common process to make Virgin
Coconut Oil is a fermentation process without heating andadding harmful chemicals compound. This process makes
Virgin Coconut Oil has a good quality (Suryani, et al., 2014).
Lactobacillus plantarum, Lactobacullus thermobacter,
Corineaebacterium bovis, Corineaebacterium xerocis andMicrococcus luteus were reported in coconut milk (Suryani,
et al., 2014). One of the functions of Lactic Acid Bacteria isbio-preservative. in this study, was conducted to determine
the species of Lactic Acid Bacteria in Virgin Coconut Oil as
bio-preservative.

MATERIALS AND METHODS
Sample Collection—An advanced commercial Virgin
Coconut Oil sample which made in west Sumatra wascollected randomly in Padang city.

Phylogenetic
Tree
Construction—Whole-genome
sequencing obtained were then assembled into contiguous
sequences (contigs) using DNAStar, about over a hundred
genomic contigs were edited use BioEdit and aligned using
the Bioedit-ClustalW Multiple Alignment. Phylogenetics tree
was made using Mega7 Construct/ Test Neighbor-Join Treetaking 1000 as bootstrap value.

Isolation and Characterization of Lactic Acid Bacteria
from Virgin Coconut Oil (VCO)—Serial dilution techniqueuntil 10-8 was used for isolation Lactic Acid Bacteria in MRS
broth (Becton, Dickinson and Company - USA), plate on
MRS agar at 37ÛC for 24 hours in anaerobic condition. Lactic
Acid Bacteria (LAB) was grown on MRS broth and MRS
agar and incubated at 37°C for 24 hours (Syukur, Fachrur,
Jamsari, & Endang, 2014). One colony was selected
randomly based on the shape, size, and color of the colony.
Isolate was stored at -80°in MRS Broth containing 20%
glycerol (v/v).

RESULTS AND DISCUSSION
Total Colonies of Lactic Acid Bacteria (BAL)—MRS Agarand MRS Broth are the media that used in this method. The
total Colonies of Lactic Acid Bacteria in this sample is 9.4 x
108 CFU/mL. The macroscopic colony of BAL showed whitesmall rounded colonies, shiny, slippery and convex edges.

Molecular Identification of Isolates
DNA Isolation—Bacteria were grown in MRS Broth at37ÛC for 18 hours. The cells were harvested, and to extract
bacterial genome were used Extrap Soil DNA Kit Plus Ver.2.
(Nintetsu Sumikin Kankyo Kabushiki Gaisya, Japan)
according to the manufacturer’s protocol. Electrophoresis
was performed on 1% agarose gel in TAE 1X buffer andphotographed under UV light.

Isolation Genomic DNA and 16S Ribosomal (rRNA) gene
amplification with PCR—One of the general methods used to
identify Lactic Acid Bacteria is the analysis of 16S rRNA
sequence. The amplification’s result showed the PCR productwas 1500 bp (Figure 2). PCR used 27F and 1525R universal
primer of 16s rRNA locus.

16S rRNA Sequencing—The 16S rRNA gene fragment of~1.5 kb was amplified by using a pair of universal primers 27
F: (5’- GAGTTTGATCCTGGCTAG-3’) and 1525 R: (5’AGAAAGGAGGTGATCCAGCC-3’). Polymerase chain
reactions (PCR) were carried out in a Fast reaction Tube
(Applied Biosystems, USA) in a total volume of 25 ȝl
containing 12.5 ȝl 2 × Green Master Mix PCR (Promega,120

Proceedings of International Symposium on Animal Production and Conservation for Sustainable Development 2018
UGSAS-GU & BWEL Joint Poster Session on Agricultural and Basin Water Environmental Sciences 2018: P20

M

C(Pb) Binding Ability; Isolated From Dadih In Sijunjung,West Sumatera, Indonesia. Research Journal of
Pharmaceutical, Biological and Chemical Sciences, 871–
876.

A B

3000bpVriesa, M. C., Vaughan, E. E., Kleerebezem, M., & Vos, a.
W. (2006). Lactobacillus plantarum—survival, functional
and potential probiotic properties in the human intestinal
tract. International Dairy Journal, 1018–1028.

1500bp1000bp

Zakaria, Z. A., Rofiee, M. S., Somchit, M. N., Zuraini, A.,
Sulaiman, M. R., Teh, L. K., . . . and Long, K. (2011).
Hepatoprotective Activity of Dried- and FermentedProcessed Virgin Coconut Oil. Evidence BasedComplement Alternat Medicine, 1-8.

Fig. 1: Electrophoresis PCR Product using 27F and
1525R universal primers of 16S rRNA, M: Marker, C:
Positive Control, A and B: Isolates from Virgin Coconut
Oil
Phylogenetic Tree Construction—The sequencer was used
DNA Sequence used Multi – capillary DNA Sequencer "ABIPrism 3100 / 3130 Genetic Analyzer” (Gifu University,
Japan). The sequence from both directions, reverse andforward is contiq by BioEdit and aligned using the BioeditClustalW Multiple Alignment. The sequence was analyzed
with data GeneBank NCBI BLAST program. Genomic
analysis 16S rRNA gene sequencing confirmed the isolate
showed that 99% is similar to Lactobacillus sakei and
Lactobacillus plantarum.
The phylogenetic tree is formed in order to determine theclothes of species based on their similarities and genetic
differences with MEGA7 Construct/ Test Neighbor-Join Treetaking 1000 as bootstrap value.

REFERENCES
H Ibrahim, A., Haibo, L., Sawsan, S. A.-R., Aman, S. A.,
Omar, A. A.-H., Xiaobo, X., . . . J, a. D. (2017).
Angiogenic and wound healing potency of fermented
virgin coconut oil: in vitro and in vivo studies. American
Journal of Translational Research, 4936-4944.
Kamdem, S. S., Guerzoni, M. E., Baranyi, J., & Pin, a. C.
(2008). Effect of capric, lauric and Į-linolenic acids on thedivision time distributions of single cells of
Staphylococcus aureus. International Journal of Food
Microbiology, 122-128.
Khoramnia, A., Afshin, E., Raheleh, G., Zahra, A., & and OiMing, L. (2013). Improvement of Medium Chain Fatty
Acid Content and Antimicrobial Activity of Coconut Oil
via Solid-State Fermentation Using a MalaysianGeotrichum candidum. BioMed Research International, 19.
Suryani, Abdi, D., Yunazar, M., Syukri, A., Edison, M., &and Nasril, N. (2014). Antimicrobial and Antifungal
Activity of Lactic Acid Bacteria Isolated from Coconut
Milk Fermentation. Research Journal of Pharmaceutical,
Biological and Chemical Sciences, 1587-1595.
Syukur, S., Fachrur, R., Jamsari, & Endang, P. (2014).
Isolation, DNA Molecular Characterization Of Lactic Acid
Bacteria Potential For Probiotic, Antimicrobial, And Lead

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UGSAS-GU & BWEL Joint Poster Session on Agricultural and Basin Water Environmental Sciences 2018: P21

The effect of Japanese persimmon (Diospyros kaki) extract on theprevention of sarcopenia
Nayla Majeda Alfarafisa1, Kohiji Kitaguchi2 and Tomio Yabe2,3
1. The United Graduate School of Agricultural Science, Gifu University
2. Faculty of Applied Biological Sciences, Gifu University
3. G-CHAIN, Gifu University
extraction and fractionation steps have been done in order to isolate
water-soluble bioactive compound from Japanese persimmon which
may has an effect on muscle atrophy pathophysiological condition.

INTRODUCTION
Sarcopenia is an age-related syndrome which characterized byprogressive loss of mass, strength, and function of skeletal muscle(Budui et al., 2015). People with sarcopenia syndrome usuallyexperienced a progressive and irreversible loss of body weight,
followed by muscle wasting or muscular atrophy, decreased muscle
stregth, and in severe cases, patient can experienced permanent
paralysis and even death. Sarcopenia is a multifactorial syndrome.
Several studies suggested that this syndrome can be caused by alteredendocrin function, genetic influence, neurodegeneration, disuse,nutrition, oxidative stress, and chronic inflammation (Kim & Choi,2013). As of now, the exact underlying mechanisms andpathophysiology of sarcopenia remain unclear. At molecular level, theloss of skeletal muscle fibers are resulted from the disturbance ofmuscle protein turnover, in which muscle protein breakdown rates
exceeds muscle protein synthesis (Sandri, 2013). The activation of thePI3K/Akt pathway in skeletal muscle cells is still reliable as a main
regulator in muscle protein synthesis through the phosphorylation ofmTOR which causes the activation of p70S6 kinase and 4E-BPI,
promoting protein translation (Chopard et. al., 2009). On the other
hand, ubiquitin-proteasome pathway appears as a main catalyst for the
majority of protein breakdown in mammalian cells, including muscle
cells (Lecker et. al., 1999). This pathway involves the sequential
action of three ubiquitin enzymes: E1 ubiquitin-activating enzyme, E2ubiquitin-conjugating enzyme, and E3 ubiquitin-protein ligase enzyme(Chopard et al., 2009).
Nowadays, the use of natural bioactive compound from plant extract
as therapeutic agent had gained much attention. Various botanicalcompound proved to be effective in maintaining muscle health. Kim etal. (2012) stated that the addition of flavonoid from Korean C.
aurantium L. can prevent muscular atrophy caused by LPS in L6
skeletal muscle cells through the regulation of inflammatory activity.
Flavonoid from Korean Citrus aurantium L. has a role as antiinflammatory agent that can regulate the expression of inflammatory
mediators and affecting NF-ț% signaling pathway. On the other hand,
Wang et al. (2014) also found out that resveratrol, biochemicalcompound in grape, can reverse C2C12 muscle cell atrophy caused by
TNF-ĮE\SUHYHQWLQJPXVFOHSURWHLQGHJUDGDWLRQDQGSURPRWHPXVFOH
protein sythesis through IGF1-Akt-mTOR signaling pathway. These
two papers indicate the potential use of the botanicals compound forthe prevention and treatment of muscle function.
Japanese persimmon (kaki fruit persimmon) (Diospyros kaki) is oneof a well known and highly distributed fruits around the world,
especially among east asian countries including Japan. This fruit
enriched with many bioactive compounds that have beneficial effecton human health owing to their ability to prevent various ailments(Yaqub et al., 2016). Bioactive compounds which prevalence inpersimmon fruit such as phenolics, flavonoids, tannins,proanthocyanidins, terpenoids, carotenoids, and vitamins. It issuggested that bioactive compound in Japanese persimmon also had
an effect to pathological condition of sarcopenia.
This study aims to investigate the effect of water solublecompounds from Japanese persimmon extract on skeletal muscle
atrophy prevention through muscle protein turnover activity(downstream part), specifically in related to chronic inflammation(upstream part) as the main cause. Muscle protein turnover will beanalyzed by evaluating the downstream of PI3K/Akt signal molecules(S6K1, mTOR, and 4E-BP1) and the activity of muscle-specific E3
ubiquitin-ligase enzymes (MAFbx and MuRF-1). On the other hand,the effect of Japanese persimmon-derived water-soluble extract on theregulation of the inflammatory pathway will be analyzed byexamining the expression of pro-inflammatory cytokine (iNOS, COX2, TNF-ĮDQG,/-6) as well as inflammatory signal molecules activity(I-NB, NF-NB and MAPKs). Currently in this research progress, theMATERIALS AND METHODS
Materials
Diospyros kaki Nishimura cultivar was provided by Gifu Prefectural
Agricultural Technology Center (Gifu, Japan) and used as a sample.
(±)-6-Hydroxy-2,5,7,8-tetramethylchromane-2-carboxylic acid andTrolox (Sigma-Aldrich, St. Lois, USA) were used as a standard formeasuring antioxidant activity, and 1,1-Diphenyl-2-picrylhydrazyl(DPPH: Wako Pure Chemical Industry, Osaka, Japan) was used as astable free radical which turns yellow when scavenged.
Japanese persimmon extraction
After washed and properly handled, the raw material of Japanesepersimmons was freezed at –30ºC until it was ready to process. The
samples were completely dryed by lyophylization methods. Afterexclude the calyx and seeds, the remaining parts of the samples were
weighted and recorded as dry weight. The samples then crushed usingmortar in order to improves the efficiency of extraction by increasing
their surface area. Powdered samples (42.5 g) were extracted in 420
mL of 98% aqueous ethanol for 30 min in boiled condition. After
three cycles of extractions, the extract was mixed combined andfiltered using a Buchner funnel. The solvents were vaporized using
rotary evaporator and dried up using lyophylization methods. All
crude extract was kept on –30ºC until further analysis.
Japanese persimmon fractionation
Japanese persimmon crude extract was fractionated using different
concentrations of ethanol (25, 50, and 75%). The extracts (5.00 g)
were dissolved in each solvent (50 mL) and homogenized using
magnetic stirrer for 1 h at room temperature. When the crude extract
completely dissolved in ethanol solvent, the samples were fractionatedagain using butanol (1:1) solvent until two different phases wereobtained. Solvent were vaporized using rotary evaporator and dried up
using lyophylization methods. All samples were kept on –30ºC until
further analysis.
DPPH radical scavenging activity
The free radical scavenging activity of the extract was measured interms of hydrogen donating or radical scavenging ability using thestable free radical DPPH. Gradient concentration (3.00-11.00 mg/mL)of Japanese persimmon fraction solutions in water was prepared. On
the other hand, trolox solution was prepared by dissolving trolox inethanol. Gradient concentration of trolox were used as a standard
curve. 30L of Japanese persimmon fraction and trolox solutions wereadded to 96-well plate, followed by 240L of methanol. Water wasused as a blank. 1mM solution of DPPH in ethanol was prepared andadded to each well of the samples. After 30 min of incubation in dark
condition, the absorbance was measured at 540nm. The assay was
performed in triplicate. Percentage of inhibition of free radical DPPH
was calculated by using equation bellows:   ሺΨሻሺ െ ሻൌͳͲͲ
Ab = Absorbance of the blank
As = Absorbance of the sample

122Proceedings of International Symposium on Animal Production and Conservation for Sustainable Development 2018
UGSAS-GU & BWEL Joint Poster Session on Agricultural and Basin Water Environmental Sciences 2018: P21

investigation after extraction and fractionation step can becathegorized as:
1. Qualitative chemical analysis: including the determination of
bioactive compound chemical properties and their nature
constituents
2. Quantitative chemical analysis: including the determination of
the purity and concentration of an isolated substance
3. Bioassay: including the determination of the biological or
pharmacological activity of substances in dosage dependent
manners.

RESULTS AND DISCUSSION
Japanese persimmon extraction
The main focus on this research is Japanese persimmon-derived
water-soluble bioactive compounds. In compared with lipid soluble
bioactive compounds, water soluble bioactive compounds had several
advantages. Recent study suggested that hydrophilic compounds had
more simple mechanism of absorption in digestive tract (Rein et al.,2013). In addition, researches that focused on this bioactive compoundare still rarely to be found. Table 1 showed an extraction yield of hot
ethanol extraction from Japanese persimmon. Theoretically, thequantity of extractive yield was directly affected by the type of
extraction methods/solvents and their extraction efficiency (Sharma &Cannoo, 2016). In this research, we conducted three cycles for eachextraction step. Relatively high number of yield percentage fromextraction step indicated the method of extraction which was chosen
can be said sufficiently effective to isolate water soluble bioactive
compound from Japanese persimmon. Several studies using hazelnut
also suggested that hot ethanol extraction (80o C, 30 min) was themost prominent method for phenolic extraction because it showed thehighest extraction yield and total phenolic content (Shahidi et al.,2007).
Table 1. Japanese persimmon extraction yield
Dry Sample (g)Crude Extract (g)Yield (%)
42.5

27.8

Antioxidant activity
Until now, we still doing antioxidant activity analysis using DPPH
method to evaluate the biological activity on each fraction. For the
future plan, we propose to carry out all the investigation methods
mentioned above to evaluate all fraction that have been obtained. Asfrom the investigation results, we can conclude about which fraction
have the highest potential on biological activity and which fraction
have the potential effect to overcome the pathophysiological conditionof muscular atrophy.

REFERENCES
Budui, S. L., Rossi, A. P., & Zamboni, M. (2015). The pathogenetic
bases of sarcopenia. Clin. Cases Miner. Bone Metab., 12, 22-26.
Chopard, A., Hillock, S., & Jasmin, B. J. (2009). Molecular eventsand signaling pathways involved in skeletal muscle disuseinduced atrophy and the impact of countermeasures. J. Cell. Mol.
Med., 13, 3032-3050.
Kim, J.-A., Park, H.-S., Kang, S.-R., Park, K.-I., Lee, D.-H.,
Nagappan, A., et al. (2012). Suppressive Effect of Flavonoids
from Korean Citrus aurantium L. on the Expression of
Inflammatory Mediators in L6 Skeletal Muscle Cells. Phytother.
Res., 26, 1904-1912.
Kim, T. N., & Choi, K. M. (2013). Sarcopenia: definition,
epidemiology, and pathophysiology. J. Bone Metab., 10, 1-10.
Lecker, S. H., Solomon, V., Mitch, W. E., & Goldberg, A. L. (1999).
Muscle protein breakdown and the critical role of the ubiquitinproteasome pathway in normal and disease states. J. Nutr., 129,227-237.
Rein, M. J., Renouf, M., CruzǦHernandez, C., ActisǦGoretta, L.,
Thakkar, S. K., & Pinto, M. D. (2013). Bioavailability ofbioactive food compounds: a challenging journey to bioefficacy.
Br. J. Clin. Pharmacol, 75, 588-602.
Sandri, M. (2013). Protein breakdown in muscle wasting: role ofautophagy-lysosome and ubiquitin-proteasome. Int. J. Biochem.
Cell Biol., 45, 2121-2129.
Shahidi, F., Alasalvar, C., & Liyana-Pathirana, C. M. (2007).
Antioxidant phytochemicals in hazelnut kernel (Corylus avellana
L.) and hazelnut byproducts. J. Agric. Food Chem., 55, 12121220.
Sharma, A., & Cannoo, D. S. (2016). A comparative study of effects
of extraction solvents/techniques on percentage yield,
polyphenolic composition, and antioxidant potential of various
extracts obtained from stems of Nepeta leucophylla: RP-HPLCDAD assessment of its polyhenolic constituents. J. Food
Biochem., 41, e12337
Visht, S., & Chaturvedi, S. (2012). Isolation of Natural Products.
Journal of Current Pharma Research, 2,584-599.
Wang, D.-T., Yin, Y., Yang, Y.-J., Pei-Jia, Shi, Y., Lu, L., et al.
(2014). Resveratrol prevents TNF-Į-induced muscle atrophy via
regulation of Akt/mTOR/FoxO1 signaling in C2C12 myotubes.
International Immunopharmacology, 19, 206-213.
Yaqub, S., Farooq, U., Shafi, A., Akram, K., Murtaza, M. A., Kausar,
T., Siddique, F. (2016). Chemistry and Functionality of Bioactive
Compounds Present in Persimmon. J. Chem. 2016. 3424025

65.3

Japanese persimmon fractionation
After underwent an extraction step, Japanese persimmon crude
extract was fractionated using different concentration of ethanol.
There are 25, 50, and 75% (v/v) of ethanol solutons which were used
as a solvent for fractionation. Table 2 showed total yield fromJapanese perimmon fractionation using 25% of ethanol. Afterdissolved in 25% of ethanol, the crude extract of Japanese persimmon
were completely dissolved in the solvent, indicating that 25% of
ethanol was not an effective solvent for separating bioactive
compound. Two different phases were obtained after the solution was
extracted again using 1-butanol. In total, from 5.00 g of sample, wehave obtained 57.0% of butanol phase, and 35.0% of 25% ethanol
phase.
Table 2. Total yield from 25% ethanol fractionation
and butanol separation
Crude Extract
25% Ethanol Phase
Butanol Phase

5.00 g1.75 g2.85 gFractionation using 50% of ethanol also dissolved Japanesepersimmon crude extract completely. Therefore, the sample needs tobe extracted again using 1-butanol to separate bioactive compound
based on polarity. In summary, 50% ethanol fractionation and butanol
separation method can obtained 18.4% of butanol phase and 70.6% of
50% ethanol phase (Table 3).
Table 3. Total yield from 50% ethanol fractionation
and butanol separation
Crude Extract
50% Ethanol Phase
Butanol Phase

5.00 g3.53 g0.92 gOn the other hand, slightly different results are obtained fromfractionation using 75% ethanol. The solvent could separate Japanesepersimmon crude extract into two different phase, there are 12.8% of
filtrate phase and 86.0% of 75% ethanol phase (Table 4). In total,
fractionation of Japanese persimmon crude extract using gradedconcentration of ethanol could produce six different fractions.
Table 4. Total yield from 75% ethanol fractionation
Crude Extract
75% Ethanol Phase
Filtrate Phase

5.00 g4.30 g0.64 gAccording to Visht and Chaturvedi (2012), a number of analyticalprocedures must be carried out to evaluate the extraction andfractionation methods of plant bioactive compounds. Three types of123Proceedings of International Symposium on Animal Production and Conservation for Sustainable Development 2018
UGSAS-GU & BWEL Joint Poster Session on Agricultural and Basin Water Environmental Sciences 2018: P22

Novel synthesis of haloalkyl nucleobase substituted at 3'-overhang ofmodified small interfering RNAs to enhance RNAi activity andexonuclease resistance
Akash Chandela1, Taeko Watanabe2, Kenji Yamagishi3 and Yoshihito Ueno1,3
1.
2.
3.

The United Graduate School of Agricultural Science, Gifu University, Japan
Department of Chemical Biology and Applied Chemistry, College of Engineering, Nihon University, Japan
Course of Applied Life Science, Faculty of Applied Biological Sciences, Gifu University, JapanINTRODUCTIONMETHODSRNA interference (RNAi) is a conserved self-defense
mechanism found in most eukaryotes where small doublestranded RNA (dsRNA) triggers a series of biochemical
events and results in sequence-specific suppression of gene
expression (Elbashir et al., 2001; Fire et al., 1998). These
small interfering RNAs (siRNAs) are short (21-23 nucleotidesin length), double-stranded regulatory RNAs that contain two
signature characteristics: a two nucleotide 3’-end overhang
and a phosphorylated 5’-end (Sharp, 2001). The functional
strand, referred as guide strand, interacts directly with themulti domain AGO proteins to form RNA induced silencing
complex (RISC). The RISC recognizes messenger RNAs that
contain sequences complementary to the guide strand andcleaves the mRNA. This eventually leads to inhibition ofprotein expression of the target mRNA.
AGO protein, a key molecule, has four domains: the Nterminal (N), Piwi/Argonaute/Zwille (PAZ), middle (MID),
and P-element-induced wimpy testis (PIWI) domains(Elkayam et al., 2012; Schirle and MacRae, 2012). The PAZ
domain recognizes the 3’-end of guide strand whereas 5’-end
is recognized by MID domain. The PIWI domain possesses
endonuclease activity which helps in cleaving the mRNA. The
PAZ domain has also been reported to harbor the nucleotidesof the 3’-dangling end in its hydrophobic pocket (Lingel et al.,
2004; Ma et al., 2004), which forms basis of our research.
Preceding reports have stated that enhanced binding with
PAZ domain leads to low RNAi activity, whereas H-bonding
seems to do the opposite (Kandeel and Kitade, 2013; Somoza
et al., 2010; Valenzuela et al., 2016; Xu et al., 2015). Ourgroup has also reported several 3’-end modifications
exhibiting resistance to nuclease as well as mitigating theRNAi activity (Inada et al., 2015; Ueno et al., 2009). In orderto evaluate the effect of electrostatic interaction as well as Hbinding associated binding with PAZ domain to changesilencing activity, we selected haloalkyl nucleobase
modification at the 3’-overhang (Fig. 1).

Thermal denaturation study
The thermally induced transition of each mixture wasmonitored at 260 nm on UV-Vis spectrophotometer fitted witha temperature controller in quartz cuvettes with a path lengthof 1.0 cm and a 3.0 M duplex concentration. The sample
temperature was increased by 0.5ÛC/min.
Dual-luciferase reporter assay
Twenty-four hours before transfection, HeLa cells
expressing luciferase genes (8 x 104/mL) were transferred to a
96-well plate (100 L/well). Cells in each well weretransfected with the indicated amounts of siRNA, andLipofectamine RNAimax (1.5 L) in Opti-MEM I ReducedSerum Medium (IQYLWURJHQ  DQG LQFXEDWHG DW ÛC.
Transfection without siRNA was used as a control. After 1 h,
DMEM (50 L) containing 10% BS was added to each well,anGWKHZKROHZDVLQFXEDWHGDWÛC. After 24 h, solution ineach well was removed and the plate was incubated at -ÛC.
After more than 4 h, activity of firefly and Renilla luciferases
in cell were determined with a dual-luciferase assay system(Promega) according to a manufacturer's protocol. The results
were confirmed by at least three independent transfection
experiments.
Partial digestion of ONs by SVPD
Each ON (600 pmol) labeled with fluorescein at the 5’-end
was incubated with SVPD  XQLW  LQ D EXIIHU  ȝL)
comprising 0.1 M Tris-HCl (pH 8.0) and 20 mM MgCl2 at
37ÛC. After 0, 1, 5, 10, 30, 60, 120, or 240 min, an aliquot ȝL) of the reaction mixture was mixed with the loading
EXIIHU  ȝL), comprising Tris-borate-EDTA (TBE) buffer
and formamide. Each sample was analyzed by 20% denaturing
PAGE at room temperature for 2 h at 20 mA. The gel was
visualized by use of a Luminescent Image analyzer LAS-4000(Fujifilm).
Molecular modeling for binding affinity
PAZ domain-siRNA complex from the Protein Data Bank
(PDB); the entry 1SI3 was used for preparing the structure.
The PAZ domain-siRNA complex prepared for calculationconsists of PAZ domain (Mse224-Gln295, Val302-Arg349)
and a 9-mer RNA (5’-CGUGACUCU-3’). Three-dimensional
data for 3’ end modified siRNA were constructed by replacing
native siRNA. The ab initio Fragment Molecular Orbital(FMO) calculations were adopted to evaluate the specificinteractions and binding affinity between PAZ domain andsiRNA. All FMO calculations were performed with the PAICS
program, where we employed the second-order Møller-Plesset(MP2) perturbation methods with the basis set of 6-31G**.

Fig.1. Structure of nucleoside analogs.
Here, in this study, we report the novel and facile synthesisof haloalkyl nucleobases whose incorporation at 3’-overhang
improve binding to the PAZ domain and lead to enhanced
silencing activity. Furthermore, these modifications also
increased resistance against exonuclease. The augmented
activity provided substantial evidence for correlating PAZ
domain binding to the silencing activity with the association ofhydrophobic moieties.

124

Proceedings of International Symposium on Animal Production and Conservation for Sustainable Development 2018
UGSAS-GU & BWEL Joint Poster Session on Agricultural and Basin Water Environmental Sciences 2018: P22

DISCUSSIONRESULTS
Synthesis

This study was directed to exploit the interaction of 3’overhang of siRNA with the PAZ domain to mitigate theRNAi activity. And, with the incorporation of hydrophobic
nucleobases, the binding as well as activity was found toincrease in a similar trend. Also, the binding studies showed
improved interaction with basic residues in the PAZ pocket.
All these observations strongly support our hypothesis foraugmented silencing activity with enhanced electrostaticinteraction between the overhang and the PAZ domain. In
addition, these modifications also proved beneficial in
improved resistance against 3’-exonuclease, with molecularsize and flexibility of the nucleobase being supposed for sucha behavior. Hence, we could successfully report the design andsynthesis of these novel siRNAs with enhanced resistance andsilencing activity. Further studies with these moieties could
add more dynamics to the ongoing research in the field.

Scheme 1. Synthesis of phosphoramidites for haloalkyl
nucleobases.

REFERENCES

The synthetic route used to synthesize phosphoramidites
20-22 of haloalkyl analogs is shown in Scheme 1. This simple
approach was followed to eventually obtain phosphoramidites
and solid supports (Scheme 2) of haloalkyl nucleobases.

Elbashir, S.M., Lendeckel, W., Tuschl, T., 2001. RNAinterference is mediated by 21- and 22-nucleotide
RNAs. Genes Dev. 15, 188–200.
Elkayam, E., Kuhn, C., Tocilj, A., Haase, A.D., Greene, E.M.,
Hannon, G.J., 2012. The Structure of Human
Argonaute-2. Cell 150, 100–110.
Fire, A., Xu, S., Montgomery, M.K., Kostas, S.A., Driver,
S.E., Mello, C.C., 1998. Potent and specific genetic
interferencebydouble-strandedRNA
in
Caenorhabditis elegans. Nature 391, 806–811.
Inada, N., Nakamoto, K., Yokogawa, T., Ueno, Y., 2015.
Synthesis of small interfering RNAs containing acetaltype nucleoside analogs at their 3'-ends and analysis of
their silencing activity and their ability to bind to the
Argonaute2 PAZ domain. Eur. J. Med. Chem. 103,
460–472.
Kandeel, M., Kitade, Y., 2013. In silico molecular docking
analysis of the human Argonaute 2 PAZ domain reveals
insights into RNA interference. J. Comput. Aided. Mol.
Des. 27, 605–614.
Lingel, A., Simon, B., Izaurralde, E., Sattler, M., 2004.
Nucleic acid 3'-end recognition by the Argonaute2 PAZ
domain. Nat. Struct. Mol. Biol. 11, 576–577.
Ma, J.B., Ye, K., Patel, D.J., 2004. Structural basis foroverhang-specific small interfering RNA recognition by
the PAZ domain. Nature 429, 318–322.
Schirle, N.T., MacRae, I.J., 2012. The Crystal Structure of
Human Argonaute2. Science (80-. ). 336, 1037–1040.
Sharp, P. a, 2001. RNA interference--2001. Genes Dev 15,
485–490.
Ueno, Y., Watanabe, Y., Shibata, A., Yoshikawa, K., Takano,T., Kohara, M., Kitade, Y., 2009. Synthesis of nucleaseresistant siRNAs possessing universal overhangs.
Bioorganic Med. Chem. 17, 1974–1981.
Valenzuela, R.A.P., Onizuka, K., Ball-Jones, A.A., Hu, T.,
6XWHU 65 %HDO 3$  *XLGH 6WUDQG ƍ-End
Modifications
Regulate
siRNA
Specificity.
ChemBioChem 17, 2340–2345.
Xu, L., Wang, X., He, H., Zhou, J., Li, X., Ma, H., Li, Z.,
Zeng, Y., Shao, R., Cen, S., Wang, Y., 2015. StructureEDVHGGHVLJQRIQRYHOFKHPLFDO PRGLILFDWLRQRIWKHƍoverhang for optimization of short interfering RNA
performance. Biochemistry 54, 1268–1277.

Scheme 2. CPG-solid support synthesis
Oligonucleotide Synthesis and Thermal stability of siRNAs
Oligonucleotides (ONs) were synthesized by standard
phospharamidite method. siRNAs 1-4 were prepared with thesynthesized ONs to incorporate modification. All modified
siRNAs exhibited slightly diminished thermal stability.
Melting temperature (Tm) and change in melting temperature
ZLWKUHVSHFWWRQDWLYH ǻTm) has been indicated in Table 1.
Table 1. Sequences of siRNAs, Tm values and gene silencing

Silencing activities of siRNAs and binding affinity
Trichloroethyl modified siRNA yielded best silencing
activity and also, best binding to PAZ domain. Also, otheranalogues showed activity corresponding to their binding
interaction (Table 1).
Nuclease resistance3’-exonuclease resistance was found to be best sustainedwith tribromoethyl analogue modified ON. Other analogues
also showed resistance better than the native. The half-life
increased to around 9 folds.

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Proceedings of International Symposium on Animal Production and Conservation for Sustainable Development 2018
UGSAS-GU & BWEL Joint Poster Session on Agricultural and Basin Water Environmental Sciences 2018: P23

LtR, a candidate of transcriptional regulator of major virulence factor of
Dickeya dadantii 3937
Dina Istiqomah1 , Naoto Ogawa2 , Shinji Tokuyama2 and Masafumi Shimizu3
1. Science of Biological Resources, The United Graduate School of Agricultural Sciences-Gifu University, Japan
2. The United Graduate School of Agricultural Sciences-Gifu University; Faculty of Agriculture-Shizuoka University,Japan
3. The United Graduate School of Agricultural Sciences-Gifu University; Faculty of Agriculture-Gifu University, Japanreference genes i.e lpxC, rpoD and gyrB which are conserved
and stable to normalizing RT-PCR were designed (Hommais,
et al., 2011; Radonie, et al., 2003).

INTRODUCTION
The pathogenecity of D. dadantii is determined by theirsecreted molecules to colonize plant tissue. The soft rot
symptom is mainly caused by the production of pectate lyases(Pels) a plant cell wall degradative enzyme (Reverchon et al.
2013). Hence, pel genes encoding pectinases are the major
virulence genes in D. dadantii (Collimer et al. 1986; Duprey etal. 2016).
In the bacterial adaptation, there is a complex
transcriptional control system coordinating the expression ofvirulence genes ensures efficient expression. Initially, thesignals feed into the transcriptional regulatory system of thebacterial cells, then continue to lead the physiological andmorphological alterations which facilitate bacterial adaptation
and affective survival. There are some regulators potentiallyinvolved in bacterial survival under particular stressconditions.
The expression of genes is controlled by complex
regulatory systems. Several regulators are reported to regulate
pel genes in response to various signals (KdgR, Pir, PecS,
PecT, Fur, MfbR, VfmE). In addition, it was shown that thesugar catabolism regulator complex cAMP-CRP acts via a
direct mechanism as the main activator of the pel genes(Nasser 1997; Reverchon 1997). Previous study revealed that
there was a gene which involved in the expression of virulent
factors and pathogenicity of D. dadantii in negative manner,namely ltR (LacI transcriptional repressor). Analyzing thestructure and physical function of the LtR protein by its
interaction with the virulence gene is important tounderstanding the mechanism of transcriptional regulation in
biological components.

Table 1. Primers for Reverse Transcription

Reverse transcription using 2.0 l 5x PrimeScript Buffer, 0.5
l PrimeScript RT Enzyme Mix I, 1 pmol, 5 pmol and 10
pmol gene specific primers, 200 ng and 500 ng of total RNA
and RNAse free H2O until 10.0 l total volume (TaKaRa). ()RNA and (-) RT enzyme are the control. The reaction was
42ºC ; 15 minutes (Reverse Transcription Process), 85ºC ; 5second (RT Enzyme inactivation), 4ºC ~.
cDNA PCR
Reaction mixture 5.0 l of 10x Buffer ExTaq, 4.0 lof dNTP mix, 1.0 l of 10 pmol forward primer, 1.0 l of 10
pmol reverse primer, 2.0 l of reverse transcription reaction,
0.25 l of Ex Taq Enzyme and 36.75 l of dH2O (50.0 l total
volume) (TaKaRa). PCR reaction with hot start PCR, 98ºC ;10 second for denaturation, 60ºC ; 10 second for annealing,
72ºC ; 15 second for extention (40 cycles) (MyCycler;
Biorad). Agarose gel electrophoresis used 1.5% agarose gel(1x TAE buffer). 5.0 l PCR product + 5.0 l dH2O + 2 l 6x
DNA loading buffer were loaded in 100 volt for 30 minutes.
Real-Time PCR
Gene expression analysis was carried out using SYBRGreen I and run in Roche Lightcycler 480 (Bio-Rad).
Gel Shift Assay
To study the function of ltR, we conducted someexperiments including cloning of the gene, express the protein
and then purify the protein. By electrophoretic mobility shift
assay we analyzed the binding of LtR protein with promoter
region of related virulence genes of D. dadantii (pelA, pelEand pelD) including the adjacent gene next to ltR (lfaA gene).
The reaction mixture consist of 4.3 l sucrose, 4.0 l binding
buffer, 1.5 l BSA, 1 l of 40 pg labeled promoter DNA ofvirulence and adjacent genes, 0.2 mM cAMP, 0.5 g poly d(Ic) and 1 g protein (Ryu et al. 1995). Electrophoresis method
using 5% BIS-polyacrylamide gel with 0.5 TBE run in 90 volt

MATERIALS AND METHODS
D. dadantii strain and culture
D.dadantii 3937 No. 253 (wild type) was grown in LB
Agar and LB liquid medium (10 g/L bacto-tryptone, 5 g/L
bacto-yeast, 10 g/L NaCl (1.5% agar in addition for LB
Agar)) ; M63 medium pH 7.0 (120 g/L (NH4)2.SO4, 136 g/L
KH2PO4, 5 mg/L FeSO4.7H2O (0.2% glycerol and 1% potato
extract in addition)) ; M63 medium pH 7.0 + 0.2% glycerol +0.2% Polygalacturonic Acid (PGA) + 1% potato extract,incubation at 28ºC for ± 24 hours.
RNA Extraction
The liquid culture of D. dadantii 3937 which have 0.30.5 absorbance in OD600 were extracted by Qiagen RNAEasy
Mini Kit. The RNA concentration measured in A260, A280
and A320. RNA Electrophoresis using 1.2% agarose (2 ml 10x
FA gel buffer+ 17.64 ml RNAse free dH2O + 360 l 37%
formaldehyde), 1 l 5x RNA loading buffer + 4 l RNA wereload in 100 volt, 30 minutes.
Reverse Transcription
The primers which amplify the candidate genes which
could regulate the virulence genes (ltR) and the regulated
virulence genes (pelA, pelE and pelD) and also other genes or126

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UGSAS-GU & BWEL Joint Poster Session on Agricultural and Basin Water Environmental Sciences 2018: P23

for 90 minutes. Blotting was carried out in 0.4 A for 30
minutes.

transcription enzyme (o), while the non transcription enzyme
lane, tha bands are not appear (x). This means the reverse
transcription process was succesful (Fig.2.).

RESULTS AND DISCUSSION
The growth ability of culture of bacteria in threedifferent medium were different (Fig.2.). The culture weregrown rapidly in LB medium, M63 medium pH 7.0 + 0.2%
glycerol + 0.2% Polygalacturonic Acid (PGA) + 1% potato
extract, and M63 medium pH 7.0+ 0.2% glycerol+ 1% potato
extract respectively. The addition of PGA in M63 medium
could increase the exponential phase of bacterial growth. PGA
induces the synthesis of pectinases, which are the essential
virulence factors of D. dadantii.

Fig. 2: cDNA PCR of ltR, pel genes (pelAED) and reference
genes (lpxC, rpoD and gyrB).
Further experiments to understand the ability of LtR
protein (LtR and LtRplus) bind specifc region of promoter
DNA, the gel shift assay was conducted using the method of
Ryu et al. which was applied for FruR, a member of LacI
family repressor protein.

Fig. 1: Growth scale of D. dadantii in three different
medium. A. LB medium; B. M63 medium pH 7.0+ 0.2%
glycerol+ 1% potato extract; C. M63 medium pH 7.0 +
0.2% glycerol + 0.2% Polygalacturonic Acid (PGA) + 1%
potato extract.
Fig. 3: Gel Shift Assay of the 300 bp of pelA promoter, 276
bp of pelE promoter, 251 bp pelD promoter and 302 bp oflfaA promoter with LtR and LtRplus protein. Method
provide by Ryu et al., 1995 with small modifications.

To detect the target genes expressed at low levels,
PCR was performed on cDNA from at high sensitivity
conditions using high purity of 200 ng total RNA extracted(Table 2.). From all RNA extracted, the cDNA provides only
using RNA extracted from M63 medium pH 7.0 + 0.2%
glycerol + 0.2% Polygalacturonic Acid (PGA) + 1% potato
extract. (Fig.2.). We assumed that this is due to the PGA andpotato extract induce the expression more effective than theother medium. The synthesis of pel genes depend on

The result showed that we got enough clear shift bands inpelA with two kind proteins as well. But in pelE and pelD thebands rather smear. Also for the adjacent gene, lfaA seems
there is no binding, but the control negative section shows a
migration of free DNA than the targeted section. This due tothe affinity binding by LtR is different towards four promoter
region. Although there still some tricky situation, we assume
that the results indicated that LtR protein has a potential to bea transcriptional regulator for the three genes.

environmental or metabolic signals, such as thepresence of pectic compounds (Jiang et al. 2016).
Table 2. Concentration of RNA were used to reverse
transcription reaction.

REFERENCES
Collmer, A., and Keen, N. T. (1986). The Role of Pectic
Enzymes in Plant Pathogenesis. Annual Review of
Phytopathology 24:383–409
Jiang, X., Zghidi-Abouzid, O., Oger-Desfeux, C., Hommais,F., Greliche, N., Muskhelishvili, G., Nasser, W., andReverchon, S. (2016). Global Transcriptional Response of
Dickeya dadantii to Environmental Stimuli Relevant to the
Plant Infection. Environmental Microbiology 00(00): 00–00.
Ryu, S., Ramsier, T.M., Michotey, V., Saier, M.H.Jr., andGarges, S. 1995. Effect the FruR Regulator on Transcriptionof the pts Opreon in Escherichia coli. The Journal of
Biological Chemistry. Vol. 270. No.6:2489-2496*1 and 2 were extracted from LB medium; no.3 extracted from
M63 medium pH 7.0+ 0.2% glycerol+ 1% potatoextract andno.4 extracted from M63 medium pH 7.0 + 0.2% glycerol +0.2% Polygalacturonic Acid (PGA) + 1% potato extract.
The particular importance is the appearance of thetargete genes, i.e. ltR, pel genes (pelAED) and reference genes(lpxC, rpoD and gyrB, because, under standard agarose gelelectrophoresis conditions, this complex has the same
migration rate as the amplification product. Morover, thebands are appear only in targete genes with reverse

127Proceedings of International Symposium on Animal Production and Conservation for Sustainable Development 2018
UGSAS-GU & BWEL Joint Poster Session on Agricultural and Basin Water Environmental Sciences 2018: P24

Effect of human activity on ecology of long-tailed macaque (Macaca
fascicularis) in One Ecotourism Site, Indonesia
Dwi Nur Setyowati, 1, Kanthi Arum Widayati1 and Yamato Tsuji2
1. Department of Biology, Bogor Agricultural University
2. Primate Research Institute, Kyoto University
Interaction between the macaque and tourist. We employed ad
libitum sampling to record interactions between the macaques
and tourists. We defined the interaction when at least one
macaque and one human oriented toward each other andexchange behavior (Fuentes et al. 2008). We classified theinteraction into provisioning and four levels of aggression;Level 0 (The macaques showed submissive behavior or no
responses to tourist), Level 1 (The macaques approached toand/or taught tourists, but not caused injury), Level 2 (Themacaques demonstrated threatening or frightening behavior
with short distance (< 3 m), included alarming, showing
canine and lifting their tails) and Level 3 (The macaques
showed aggressive behavior, including bitting, chasing,stealing and damaged tourists’ belongings).

INTRODUCTION
Majority of temporal changes in primate behavior isaffected by differences in dietary availability, quality, anddistribution (Hanya 2004). The same holds for provisioned
primates (Sha and Hanya 2013b; Ménard et al. 2013). For
instance, the number of tourists (and amount of provisioning
foods) changes seasonally, which in turn affects activity, dietcomposition, and degree of aggressive behavior of themonkeys; semi-provisioned baboons (Papio cynocephalus),
spent less time for feeding and more for resting than wild
troop (Altmann and Muruthi 1988). Semi-provisioned Barbary
macaques (Macaca sylvanus) spent less time for feeding andmoving, and more for resting, while wild troops spent moretime for foraging and moving, and less for resting. And, mean
daily travel distance of the former was significantly shorter
than the latter (El Alami et al. 2012).
If primates rely much on provisioning foods, they would
adjust their activities in response to tourists' activity in given
time. In food scarce season, for example, monkey-humanconflicts might become severer than food rich season. This
study examined effects of seasonal variation in provisioningon 1) activity budgets, 2) dietary composition, 3) daily travel
distances, and 4) degree of aggressive behavior of long-tailed
macaque (Macaca fascicularis) in Pangandaran Nature
Reserve, West Java, Indonesia. Specifically, we comparedmacaque behavior among three different types of days inwhich number of tourists seemed different: 1) weekday, 2)
weekend, and 3) Ramadhan, a month of fasting.
We conducted observation on weekdays (between Monday
and Friday), weekends (Saturday and Sunday) and Ramadhan
to compare the effects of tourist number on activity budget,dietary composition, daily travel distances, and level ofinteraction with tourists. We predicted number of tourists
influenced the difference feeding strategy, in weekend movingof long-tailed macaque shorter then weekday and Ramadhan,but more feeding time. Equally, on weekend the macaquespent most feeding time on provisioned than natural food. On
weekday, long-tailed showed more aggressive behavior
toward tourists as amount of provisioned food decreased.
While on Ramadhan the daily distance of M. fascicularis waslarger and approaching urban area to obtain more provisioned
food.

Travel distance. During the behavioral observation, werecorded own locations (represented as location of subject
troop) by a handy-type GPS every 10 minutes, from which weestimated the travel distance (m) of the macaques.

RESULTS AND DISCUSSION
Activity Budget. The highest activity was resting (32-36%,
across the day types), followed by moving (27-31%) andsocial behavior (22-25%). During the study, all the activities,
except for moving, of the macaques were relatively stable
during the study. Number of tourists affected percentages ofsome activities; when the number of tourists increased,
percentage of feeding increased, while of human junkhandling activities decreased. It would be speculated that themacaques increased their foraging efforts to obtain more
provisioning foods, and as consequently, time spent for junkhandling (which is considered as searching of provisioning
foods). Previous studies have showed that provisioned longtailed macaques spent much time for moving and feeding, andless time for resting (Son 2004; Hambali et al. 2012).
Diet Activities. Across the day types, the macaques fed onprovisioned foods (17-22%), leaves (27-41%), and fruits andseeds (29-41%). There were no significant differences inpercentage among the day types. However we found thatpercentage of provisioned foods increased with number oftourists, while percentage of fruits and seeds and other natural
food. This implies that tourists had potential effects on
dietary composition of the macaques. There have been several
studies found that provisioning by tourists changed the dietarycomposition of the long-tailed macaques (Sha and Hanya
2013a). Nila et al. (2014), reported that the long-tailed
macaques at Telaga Warna Bogor fed mainly on fruits andseeds on Ramadhan season, while feeding of provisioning
food decreased. The reason why the macaques spent muchtime for provisioned foods is likely due to its higher nutrition(Wheatley 1989).

MATERIALS AND METHODSThe observation was conducted in Pangandaran Nature
Reserve (PNR), Pangandaran District, West Java, Indonesia.
Subject animal of this study was one troop of long-tailed
macaques, called Pantai Barat troop.
Activity Budget. We employed instantaneous scan sampling
every 10 minute to record activity budgets of the macaques,
and recorded activity of all visible animals. The behavioral
categories of the macaques were classified into moving,
resting, feeding, social activity, human junk handling andothers.

Travel Distance. The mean travel distances of the macaques
was 578 ± 336m, and showed no significant differencesamong the day types. We found no significant relationshipwith number of tourists. As to ranging inside the home range,
however, we found unique characteristics: on weekdays the128Proceedings of International Symposium on Animal Production and Conservation for Sustainable Development 2018
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long-tailed macaques showed activities in forest, on weekend
in tourist area, and in the urban area on Ramadhan days.
Similar finding has been reported from Kuala Selangor Nature
Park; the macaques adapted to direct provisioning, the daily
activity mostly at the edge of the park which adjacent withresidential area (Hambali et al. 2012). This study confirmedthat a pattern of range use, but not travel distances, is affectedby provisioned foods.

threatening or frightening behavior (in terms of alarming,
showing canine and lifting the tail) (Level 2). Thus, degree of
dependence on provisioning in PNR would be relatively lower
than other heavy provisioned sites, such as at Padangtegal
Monkey Forest in Bali, Indonesia, and the Upper Rock Nature
Reserve in Gibraltar (Fuentes 2006). In PNR, interaction level3 occurred when tourists did not provide foods to themacaques. This implies that the degree of aggressivenessbecomes higher when availability of the provisioned foods
decreased.

Interaction Macaque-Tourist.

REFERENCESAEl Alami A, Lavieren, EV, Rachida A, Chait A. 2012.
Differences in activity budgets and diet between semiprovisioned and wild-feeding groups of the endangered
Barbary macaque (Macaca sylvanus) in the central highAtlas Mountains, Morocco. American Journal ofPrimatology 74: 210–216.
Fuentes A. 2006. Human Culture and Monkey Behavior:
Assessing the Contexts of Potential Pathogen Transmission
Between Macaques and Humans. American Journal ofPrimatology 68: 880–896.
Fuentes A, Kalchik S, Gettler L, Kwiatt A, Konecki M, JonesEngel L. 2008. Characterizing Human–MacaqueInteractions in Singapore. American Journal of Primatology
70:879–883.
Hanya G. 2004. Diet o japanese macaque troop in the
coniferous forest of Yakushima. International Journal ofPrimatology 25: 55-71.
Hambali K, Ismail A, Md-Zain BM. 2012. Daily activity
budget of long-tailed macaques (Macaca fascicularis) in
Kuala Selangor Nature Park. International Journal of Basic& Applied Sciences 12: 44-47.
Ménard N, Motsch P, Delahaye A, Saintvanne A, Le Flohic
G, Dupe S, Vallet D, Qarro M, Pierre J. 2013. Effect of
habitat quality on the ecological behaviour of a temperateliving primate: time-budget adjustments. Primates 54: 217228.
Nila S, Suryobroto B, Widayati KA. 2014. Dietary variation of
long-tailed macaques (Macaca fascicularis) in Telaga
Warna, Bogor, West Java. Hayati: Journal of Bioscience
21: 8-14.
Sha JCM., Gumert MD, Lee BPYH, Jones-Engel L, Chan S,
Fuentes A. 2009. Macaque–human interactions and thesocietal perceptions of macaques in Singapore. American
Journal of Primatology 71: 825–839.
Sha JCM, Hanya G. 2013a. Diet, activity, habitat use, andranging of two neighboring groups of food̺ enhanced
long ̺ tailed macaques (Macaca fascicularis). American
Journal of Primatology 75: 581̽592.
Sha JCM, Hanya G. 2013b.Temporal food resource correlates
to the behavior and ecology of food-enhanced long-tailed
macaques (Macaca fascicularis). Mammal Study 38: 163175.
Son VD. 2014. Time budgets of Macaca fascicularis in amangrove forest, Vietnam. Laboratory Primate Newsletter
43: 2-4.
Wheatley BP. 1989. Diet of Balinese temple monkeys,
Macaca fascicularis. Kyoto Univ Overseas Res Rep Stud
Asian non-Human Primates 7:62-75.

BC

Fig. 1: Relationships between number of tourists andtourist-macaque interactions. A (Level 1), B (Level 2),
C (Provisioning).
Majority of interactions with human were tolerant, butsometimes aggressive interactions were observed. Frequencyof the provisioning was higher in weekend, while frequencies
of other interactions were stable among the other day types.
And, degree of level 1 and level 2 increased when number oftourists increased. That is, majority of macaque-tourist
interactions were tolerant. (Fig 1). The number of tourists had
positive relationships with degree of Level 1 and Level 2interactions. Sha et al. (2009) found that two-thirds of humanmacaque interaction of M. fascicularis in Singapore occurred
when a human carried food or food cues, which was similar tothis study. However, we also found that tourists’ presence
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Current situation, research, and conservation on bonobos in Mbali(Democratic Republic of the Congo)
Yuta Shintaku
Wildlife Research Center, Kyoto University; Japan Monkey Centre

INTRODUCTION
The bonobo (Fig. 1. Pan paniscus, Primates; Hominidae) isa great ape species, and one of the closest species to humans.
Bonobos are endemic to the Democratic Republic of theCongo (DRC), and their distribution is limited to the left bankof the Congo River. The population size of the bonobos isunclear because of limited field surveys. Fruth et al. (2016)has estimated that the minimum population size is 15,000–
20,000 individuals. However, their population size isdecreasing owing to illegal hunting, deforestation, and disease.
The IUCN Red List categorizes bonobos as Endangered (EN).
In 2005, western population of bonobos was re-discoveredin the Lac Tumba Landscape, western DRC (Ignowabini2007). The estimated population density was higher than thatin other regions; thus, this western population is important forthe conservation of bonobos.
The Mbali area (Fig. 2, Territoire du Bolobo, Province du
Mai-Ndombe) is located in the Lac Tumba Landscape and is
the southwestern edge of the bonobo distribution range. Mostof the habitat in the distribution range of the bonobos is humid
tropical forest. However, the Mbali area has a much drier
forest-savanna mosaic habitat. The Mbali area is a uniqueenvironment as bonobo habitat and important to understandbonobo adaptation to drier habitats. For example, in the Mbaliarea, savanna use by bonobos for traveling and feeding wasobserved.
In the Mbali area, a local NGO, Mbou Mon Tour (MMT),
started the habituation of several groups of bonobos withsupport from the World Wide Fund for Nature (WWF) in
2005. This habituation is aimed at developing eco-tourism
focused on wild bonobos and local environment. That is, theaim is to develop the local community through conservationand sustainable use of the local environment as tourism
resources. In addition to habituation, MMT has started a local
environment conservation program through the prohibition of
hunting using guns and traps in the forest, and slash-and-burnmanagement. Conservation efforts by local NGO have beensummarized by Narat et al. (2015).

RESEARCH
Since 2015, Japanese research teams have conducted fieldsurveys of one bonobo group habituated by MMT and WWF
DRC in Mbali (named Nkala group). According to theprogress of habituation, researchers can observe bonobos frommuch shorter distances for longer time. In future, long-term
observational data will provide important information on thesocio-ecology of bonobos in drier environments.

HEALTH MONITORING
Diseases, especially those transmitted from humans to thegreat apes, are one of the important problems for theconservation. Many cases of such zoonosis have been reported(Dunay, 2018). Especially, eco-tourism results in close contact
between humans and the great apes, thereby increasing therisk of disease transmission from humans to great apes. In
many eco-tourism sites of the great apes, health conditions aremonitored.
In 2014 and 2015, respiratory disease outbreaks occurredamong the bonobos in the Mbali area. At least four individuals
died in both the years and more individuals disappeared. The
outbreak in 2015 was occurred in the Nkala group.
Grützmacher et al. (2018) reported that the causative agentswere human respiratory syncytial virus (HRSV) and
Streptococcus pneumoniae. Owing to the outbreaks, the groupsize of Nkala decreased to around 15 individuals. For theconservation of the Mbali bonobos, zoonosis monitoring isimportant. As such, since May 2016, I have conducted healthmonitoring of the bonobos based on non-invasive sampling.
During direct observations of the bonobos in the forest, I
collected feces, urine, and food remains (Fig. 3) as samples.
Using these samples, I checked human respiratory diseases,
such as Influenza virus, Rotavirus, and Streptococcus
pneumoniae, using conventional detection kits. In addition, I

Fig. 1. Young bonobos in the Mbali area (Nkala group).
Fig. 2. Location of the Mbali area.

130Proceedings of International Symposium on Animal Production and Conservation for Sustainable Development 2018
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also surveyed the intestinal parasites using the direct smear
method of fecal samples.
Based on direct observation, use of test kits, and parasite
analyses, new outbreaks were not diagnosed in the Nkala
group of bonobos. However, continuous monitoring of healthconditions is required for sustainable eco-tourism.

This work was supported by the Leading Graduate Programin Primatology and Wildlife Science, Kyoto University, andMitsui & Co. Environment Fund Activity Grant (K15-0018).

REFERENCES
Dunay, E., Apakupakul, K., Leard, S., Palmer, J. L., Deem, S.
L. (2018) Pathogen Transmission from Humans to Great
Apes is a Growing Threat to Primate Conservation.
Ecohealth 15: 148-162.
Fruth, B., Hickey, J.R., André, C., Furuichi, T., Hart, J., Hart,
T., Kuehl, H., Maisels, F., Nackoney, J., Reinartz, G., Sop,
T., Thompson, J. and Williamson, E.A. (2016) Pan
paniscus (errata version published in 2016). The IUCN Red
List of Threatened Species 2016: e.T15932A102331567.
http://dx.doi.org/10.2305/IUCN.UK.20162.RLTS.T15932A17964305.en. Downloaded on 17
September 2018.
Grützmacher, K. S. Keil, V., Metzger, S., Wittiger, L.,
Herbinger, I., Calvignac-Spencer, S., Mätz-Rensing, K.,
Haggis, O., Savary, L., Köndgen, S., Leendertz, F. H.
(2018) Human respiratory syncytial virus and Streptococcus
pneumoniae infection in wild bonobos. EcoHealth 15: 462–
466.
Inogwabini, B.-I., Matungila, B., Mbende, L., Abokome, M.,
and Tshimanga, T. (2007) Great apes in the Lake Tumba
landscape, Democratic Republic of Congo: newly described
populations. Oryx 41: 532–538.
Narat, V., Pennec, F., Ledo-Bisson, L., Bokika Ngawolo, J.
C., Dumez, R., Krife, S. (2015) Bonobo Conservation as ameans for Local Development: an Innovative Local
Initiative of Community-based Conservation in the
Democratic Republic of the Congo. Journal of Primatology
4: 127.

Fig. 3. Leaf stem eaten by the bonobo. Mucosa of the oral cavity
from this sample was tested using conventional detection kits.

ACKNOWLEDGMENTS
I would like to thank Mbou Mont Tour (Jean-Christophe
Bokika, Inoccent Leti, Claude Monghiemo Nghapo), WWF
DRC (Olivia Haggis, Charles-Albert Petre), and all local staff,
including the bonobo trackers, for support for conducting fieldwork in Mbali. I would also like to thank Dr. Raymond
Rumbuenamo, Prof. Gen’ichi Idani (Kyoto University), Dr.
Shinya Yamamoto (Kyoto University), and L ņŤŰŭŦġ
Régionale Postuniversitaire d’Aménagement et de Gestion
intégrés des Forêts et Territoires tropicaux (ERAIFT).

131

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Population dynamics of geographically isolated population of stump-tailed
macaques in Thailand - from the viewpoint of conservation
Tamaki Maruhashi1, Aru Toyoda2, Yuzuru Hamada2, and Suchinda Malaivijitnond3,4
1. Department of Human Cultures, Musashi University, Tokyo, Japan
2. Primate Research Institute, Kyoto University, Inuyama, Aichi, Japan
3. Department of Biology, Faculty of Science, Chulalongkorn University, Bangkok, Thailand
4. National Primate Research Center of Thailand, Chulalongkorn University, Saraburi, Thailand

INTRODUCTION
The stump-tailed macaque (Macaca arctoides) is a speciesof the genus Maccaca, family Cercopithecidae. This species
can be found across Asian continent, from north-eastern India
to south-western China and downward to Vietnam, Cambodia,
Myanmar, Thailand, and Peninsular Malaysia (Fooden 1990).
Internationally, this species is categorized in VULNERABLE
on The IUCN Red List of Threatened Species, and also listed
under Appendix II in CITES. The species is protected innational wildlife acts of Lao PDR, Viet Nam, Thailand, andMyanmar.
Our study site, Khao Krapuk Khao Taomor Non-Hunting
Area, is located in Phetchaburi province, the central part ofThailand. Here is the only study site of stump-tailed macaques
where long-term continuation study has been conducted basedon individual identification. Direct observation including
population census has been conducted since 2005 by Tamaki
Maruhashi (TM) and Aru Toyoda (AT). An important andurgent conservation issue here is a rapid growth in the
population and a destruction of the habitat. Historically, thispopulation was discovered in 1984 and the population size atthat time was only 22 monkeys (7 adult males, 5 adultfemales, 6 juveniles and 4 infants) (Theesucon 1988).
However, at present, population is increased up to 391
individuals. Together with the increase in population, a part oftheir habitat was recently invaded by a construction of thephotovoltaic power plant which could be a threat to thispopulation because their foraging area was shrunken. In thisposter, we report the detailed data of population census,changes in ranging areas after a habitat destruction, and thelatest activities towards the protection of this local population.

Photo. 1: Stump-tailed macaques passing across the roadin the forest of Khao Krapuk Khao Taomor Non-Hunting
Area. This photograph was taken from the same angle as
the video camera. Photo by AT.

RESULTS
Population size –Although the population was composed ofonly 22 monkeys in 1988 (Theesucon 1988), the latest
counting by AT in 2016-2017 was 391 individuals which weredivided into 5 groups (Toyoda and Malaivijitnond 2018).
Group fission - On November 10th, 2015, AT found a small
group containing only 37 individuals. According to theindividual identification list, some males and females weremembers of the Third group, thus we concluded that this small
group was separated from the Third group and named“Wngklm group”. This group size became 43 by the end of
2017.
Provisioning – Food provisioning to wildlife including
macaques is common in Thailand regarding the principle oftheir religion of Buddhism. Unexceptionally, foodprovisioning to these stump-tailed macaques by passerby wasusual Although we did not measure the amount and thefrequency of foods provided, the high nutritional fruits such as
bananas and mangoes with high amount were frequently
provided.
Habitat destruction – In 2015, northern part of their homerange was deforested and a huge solar power plant was built.
As a consequence, the bamboo forest which was one of theirmain feeding sites was cleared up.
Activities for conservation –Department of the National
Parks, Wildlife and Plant Conservation of Thailand (DNPT)
was recently actively involved in conserving these monkeys.
Thanks to their activities, signboard telling about monkeys
crossing the road has been posted, and a provisioning along
the road which can cause a traffic accident and death tomonkeys is prohibited.
Supernumerary nipples 㸫 Although the relationshipbetween a rapid increase in population and food provisioning
is not tested, a supernumerary (>2) nipples was confirmed inthis population. Of the 332 monkeys counted, the percentage
of supernumerary nipples was 22% (Toyoda andMalaivijitnond 2018).

MATERIALS AND METHODS
Khao Krapuk Khao Taomor Non-Hunting Area located at
99°44' E, 12°48' N, and the total area was about 3.5–4 km2.
There were 5 groups of stump-tailed macaques, namely Ting,
Nadam, Third, Fourth and Wngklm group. This site consisted
primarily of secondary forest, including stands of bamboo, andagricultural areas. From our long-term survey, no natural
predators to the stump-tailed macaques were observed. The
macaques also visited the adjacent areas such as temple,
cassava and pineapple plantations, and human settlements on
their daily roaming. They were occasionally fed by humans,
both locals and tourists, at the temple grounds or along theroadside.
We recorded the group activities by a video camera,
counted the number of individuals and categorized their age
and sexes. Ranging area was recorded by GPS devise(GARMIN GPSmap 62s) which was carried by observers
when followed the monkeys.

132

Proceedings of International Symposium on Animal Production and Conservation for Sustainable Development 2018
UGSAS-GU & BWEL Joint Poster Session on Agricultural and Basin Water Environmental Sciences 2018: P26

Figure. 1: Percent occurrence of supernumerary nipples in
the Ting, Nadam, Third, Fourth, and Wngklm groups(Toyoda and Malaivijitnond 2018).

Photo. 2: Stump-tailed macaques feeding a plentiful eggplant provided by local farmer. The amount of provided
foods is often excessive, which is certainly one of the major
factors in a rapid population growth. Photo by AT.

DISCUSSION
While the global trend of the population of stump-tailed
macaques is decreased, in our study site, it is reversed. Since
they have been successfully protected and a population is
rapidly grown, without natural predators, the existing habitat
might not be able to hold them. Thus, they started to forage
into and damage to the surrounding agricultural areas which
will finally lead to a conflict with humans in the vicinity.
Since this population is geographically isolated from otherconspecific populations (Malaivijitnond et al., 2005), a geneflow between populations has been impossible. Thus, if the
population is kept increasing, an inbreeding depression and a
decline in genetic diversity could be occurred.
In the future, a multifaceted perspective, including genetic
analysis, and more effort on conservation in cooperation with
DNPT will be incorporated into our study.

Photo. 3: Solar panels set in the area of photovoltaic power
plant. Due to the construction of this power plant, themonkeys lost the major ranging area of the northern part.
Photo by AT.

ACKNOWLEDGMENTS
We are grateful to Warayut Nilpaung, Chuchat Choklap, thesuperintendent of the Khao Krapuk Khao Taomor NonHunting Area. Also, to Phanlerd Inprasoet, Wanchai Inprasoet,
and Napatchaya Techaatiwatkun for providing a great deal of
support in the continued success of this fieldwork; Dr. Hiroki
Koda, Dr. Ikuma Adachi, Dr. Ikki Matsuda and Dr. Takeshi
Nishimura for their kind supports in our research. This study
was funded by the Japan Society for the Promotion of Science
KAKENHI (#16J0098 to AT), a Young Science Explorer
Grant from the National Geographic Foundation for Scienceand Exploration - Asia (#Asia-22-15 to AT), Kyoto University
Foundation (to AT), and the Cooperation Research Program of
the Wildlife Research Center, Kyoto University (#2015-Jiyuu8 to AT).

Photo. 4: Local people giving pineapples on the roadsidenext to the signboard saying “DO NOT FEED WILDANIMALS”. Photo by AT.

REFERENCES
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Malaivijitnond S, Hamada Y, Varavudhi P, Takenaka O. (2005) The
current distribution and status of macaques in Thailand Natural
History Journal of Chulalongkorn University. supplement 1:35-45.
Treesucon U (1988) A survey of stump-tailed (Macaca arctoides) in
Thailand. Natural History Bulletin of the Siam Society 36:61-70
Aru T and Malaivijitnond S (2018) The first record of dizygotic
twins in semi-wild stump-tailed macaques (Macaca arctoides) tested
using microsatellite markers and the occurrence of supernumerarynipples. Mammal Study 43(3)133The United Graduate School of Agricultural Science, Gifu University
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