Pemanfaatan Sperma Epididimis Dan Mutasi Penyebab Munculnya Warna Belang Pada Kerbau Lumpur
THE UTILIZATION OF EPIDIDYMAL SPERM AND
CAUSATIVE MUTATIONS RELATED TO SPOTTED
COAT COLOR PATTERN IN SWAMP BUFFALO
YULNAWATI
GRADUATE SCHOOL
BOGOR AGRICULTURAL UNIVERSITY
BOGOR
2014
STATEMENT OF COPYRIGHT FOR DISSERTATION AND
SOURCES OF INFORMATION *
I hereby declare that the dissertation entitled “The Utilization of
Epididymal Sperm and the Causative Mutations Related to Spotted Coat
Color Pattern in Swamp Buffalo” is true of my work under the guidance of the
supervising committee and has not been submitted in any form to any college.
Resources derived or quoted from works published and unpublished from other
writers mentioned in the text and listed in the References at the end of this
dissertation.
I hereby bestow the copyright of my papers to the Bogor Agricultural
University (IPB).
Bogor, June 2014
Yulnawati
NIM D161100041
RINGKASAN
YULNAWATI. Pemanfaatan Sperma Epididimis dan Mutasi Penyebab
Munculnya Warna Belang Pada Kerbau Lumpur. Dibimbing oleh CECE
SUMANTRI, ARIEF BOEDIONO, RONNY RACHMAN NOOR dan GÖRAN
ANDERSSON.
Kerbau belang merupakan salah satu keanekaragaman hayati Indonesia
yang termasuk dalam kelompok kerbau lumpur atau kerbau rawa (Bubalus
bubalis carabanensis). Populasi kerbau belang umumnya ditemui di sekitar
wilayah Toraja, Sulawesi Selatan. Jenis ternak ini memiliki ikatan yang sangat
erat dengan kebudayaan setempat. Kerbau belang jantan dikorbankan untuk
dijadikan sebagai persembahan pada saat upacara kematian. Rendahnya tingkat
kelahiran akibat tradisi yang tidak menginginkan terjadinya kawin alam pada
kerbau belang jantan, serta tingkat pemotongan yang tinggi akibat upacara adat,
menyebabkan populasi ternak ini menurun drastis, mendekati kepunahan.
Disertasi ini bertujuan untuk i) menginvestigasi metode reproduksi yang
dapat diterapkan untuk meningkatkan angka kelahiran kerbau belang, dan ii)
mengidentifikasi mutasi pada gen MITF sebagai penyebab munculnya variasi
warna belang. Disertasi ini terdiri dari tiga kajian yang meliputi aspek reproduksi
kerbau belang jantan dan variasi genetik penyebab munculnya warna belang pada
kerbau rawa.
Pada kajian pertama, dilakukan investigasi mengenai pengaruh warna kulit
terhadap kualitas sperma epididimis segar dan setelah thawing. Sampel sperma
epididimis dikoleksi dari 12 ekor kerbau belang yang terdiri dari 5 ekor Saleko, 4
ekor Bonga dan 3 ekor Lotong Boko, dibandingkan dengan sperma epididimis dari
5 ekor kerbau hitam. Tidak ditemukan adanya perbedaan signifikan pada setiap
parameter kualitas sperma yang diamati, baik pada kondisi sperma segar maupun
setelah thawing. Motilitas progresif sperma epididimis setelah thawing dari
kelompok Saleko, Bonga, Lotong Boko dan hitam secara berturut-turut adalah
44%, 42%, 40% dan 42%. Sementara itu, daya hidup dan keutuhan membran
plasma sperma epididimis dari keempat kelompok tersebut secara berturut-turut
adalah 64,9%; 65,2%; 62,6%; 62,7% and 64,6%; 67,1%; 64,5%; 64,1%.
Disimpulkan bahwa variasi warna kulit belang ternyata tidak memberikan
pengaruh terhadap kualitas sperma epididimis segar maupun beku.
Kajian kedua bertujuan untuk mengetahui pengaruh dua bahan pengencer
terhadap kualitas sperma epididimis setelah thawing, serta keberhasilan
kebuntingan setelah inseminasi buatan (IB) menggunakan sperma tersebut. Bahan
pengencer yang dibandingkan adalah Tris kuning telur (TEY20) versus Sitrat
kuning telur (CEY20). Hasil yang diperoleh menunjukkan kedua jenis pengencer
memiliki kemampuan yang sama dalam mempertahankan motilitas progresif,
viabilitas dan keutuhan membran plasma sperma epididimis setelah thawing.
Sebanyak 47% (7/15) kerbau betina berhasil bunting setelah diinseminasi
menggunakan sperma epididimis setelah thawing yang diencerkan dalam TEY20
dan 40% (6/15) menggunakan sperma epididimis setelah thawing yang diencerkan
dalam CEY20. Disimpulkan bahwa kedua jenis bahan pengencer, TEY20 dan
CEY20, dapat digunakan untuk mempertahankan kualitas dan fertilitas sperma
epididimis kerbau belang setelah thawing.
Dalam kajian ketiga, dilakukan penelusuran variasi genetik pada gen
microphthalmia-associated transcription factor (MITF) dalam kaitannya terhadap
kemunculan warna belang. Studi ini berhasil mengidentifikasi dua jenis mutasi
penting, yaitu mutasi nonsense yang menyebabkan terjadinya premature stop
codon pada exon 3, dan mutasi donor splice-site yang menyebabkan perpanjangan
exon 8. Akibat mutasi splice-site ini terjadinya penambahan 8 residu asam amino
yang diperkirakan terjadi langsung sebelum leucine ketiga dari leucine zipper
bHLH-Zip domain, sehingga mempengaruhi dimerisasi dan kapasitas ikatan
protein MITF dengan DNA.
Secara umum dapat disimpulkan bahwa IB menggunakan sperma epididimis
kerbau belang yang dibekukan baik dalam bahan pengencer TEY20 maupun
CEY20 dapat diaplikasikan guna meningkatkan angka kelahiran kerbau belang.
Selanjutnya, dapat dipastikan bahwa mutasi nonsense dan splice-site yang
diidentifikasi pada gen MITF merupakan faktor penyebab munculnya warna
belang pada kerbau rawa.
Kata kunci: Sperma Epididimis, Bahan Pengencer, Inseminasi Buatan, MITF,
Kerbau Belang
SUMMARY
YULNAWATI. The Utilization of Epididymal Sperm and Causative Mutations
Related to Spotted Coat Color Pattern in Swamp Buffalo. Supervised by CECE
SUMANTRI, ARIEF BOEDIONO, RONNY RACHMAN NOOR and GÖRAN
ANDERSSON.
The spotted buffalo is an Indonesian domestic buffalo, which is a variant of
the swamp buffalo (Bubalus bubalis carabanensis) type. The spotted buffalo is
mainly found in the Toraja region, South Sulawesi. Apart from its importance in
agriculture, this species is also closely tied to local spiritual beliefs. The sacrificial
offering of spotted buffalo is considered a fundamental part of funeral
ceremonies. Due to the spotted buffalo’s low birth rate of natural reproduction,
but yet high demand in funeral ceremonies, the species is close to extinction.
The objectives of this dissertation were i) to investigate the applicative
reproductive methods that are useful to increase the birth rate of spotted buffalo,
and ii) to identify the causative mutations and variations in MITF gene that are
associated with spotted coat color pattern in swamp buffalo. This dissertation
consists of three studies, two studies were related to the reproductive aspect of
spotted buffalo bulls and the third study was related to the variation of a
pigmentation gene in swamp buffalo.
In the first study we investigated if fresh and frozen-thawed epididymal
sperm quality was influenced by the buffalo’s coat color pattern. Therefore,
epididymal sperm from 12 buffaloes with spotted coat color pattern (5 Saleko, 4
Bonga and 3 Lotong Boko) were compared to sperm from 5 buffaloes with solid
coat color pattern. Results showed that the coat color pattern did not influence the
sperm quality. No significant difference was found in any of the analyzed
parameters among groups of fresh and frozen-thawed sperm. The percentage of
frozen-thawed progressive motility from Saleko, Bonga, Lotong Boko and Solid
was 44%, 42%, 40% and 42%, respectively. Moreover, the percentage of viability
and membrane integrity of frozen-thawed sperm were 64.9%; 65.2%; 62.6%;
62.7% and 64.6%; 67.1%; 64.5%; 64.1%, respectively. In conclusion, the buffalo
coat color pattern variation did not affect the quality of fresh and frozen-thawed
epididymal sperm.
The objective of the second study was to investigate the ability of
chemically defined extenders in maintaining the quality and fertility of frozenthawed epididymal sperm of spotted buffaloes. Two extenders, Tris-based egg
yolk (TEY20) and Citrate-based egg yolk (CEY20), were used in this study. The
results showed a similar progressive motility, viability and membrane integrity of
frozen-thawed epididymal sperm in both extenders. With 30 buffalo cows, the
resulting success rate of pregnancy was 47% (7/15) using frozen-thawed
epididymal sperm in TEY20 and 40% (6/15) using the one in CEY20 extender. In
conclusion, the two chemically defined extenders (TEY20 and CEY20) were
equally suitable to maintain the quality and fertility of frozen-thawed epididymal
sperm.
In the third study we identified the genetic variation in the microphthalmia
associated transcription factor (MITF) gene that is associated to the spotted coat
color pattern. We identified two important causative mutations: first, a nonsense
mutation that caused premature stop codon in exon 3 and likely removal of the
resulting mRNA via nonsense-mediated mRNA decay pathway; second, a donor
splice-site mutation that led to aberrant mRNA splicing of exon 8 of MITF
resulting in an mRNA coding for protein with eight additional amino acid
residues. Most likely the insertion of the eight amino acid residues negatively
influences dimerization and DNA binding capacity because the insertion was
located immediately before the third leucine in the leucine zipper bHLH-Zip
domain.
In conclusion, AI using frozen-thawed epididymal sperm, which was diluted
either in TEY20 or CEY20, could be applied to increase the birth rate of spotted
buffaloes. Furthermore, the nonsense and splice-site mutations in MITF gene were
strongly associated with spotted coat color pattern in swamp buffaloes.
Keywords: Epididymal Sperm, Extenders, Artificial Insemination, MITF, Spotted
Buffalo
© All Rights Reserved IPB, in 2014
Copyright Reserved
Prohibited from quoting part or all of this paper without including or citing
sources. Citations only for educational purposes, research, writing papers,
preparing reports, writing criticism, or review a matter, and citations are not
detrimental to the interests of the University.
Prohibited announced and reproduce part or all of this publication in any form
without permission from IPB.
THE UTILIZATION OF EPIDIDYMAL SPERM AND
CAUSATIVE MUTATIONS RELATED TO SPOTTED
COAT COLOR PATTERN IN SWAMP BUFFALO
YULNAWATI
Dissertation
As a requirements for Doctoral degree
at
Major of Livestock Production and Technology
GRADUATE SCHOOL
BOGOR AGRICULTURAL UNIVERSITY
BOGOR
2014
Title
Name
Student ID
: The Utilization ofEpidiymal Spenn and Causative Mutations
Related to Spotted Coat Color Pattern in Swamp Buffalo
: Yulnawati
: D161100041
Approved by
Supervision Committee
Prof Dr Ir Cece Sumantri, MAgrSc Prof AriefBoediono, DVM, PhD, PAVet (K)
Main Supervisor
Co-Supervisor
Prof Dr Ir Ronny R. Noor, MRurSc
Co-Supervisor
Prof Dr Goran Andersson
Co-Supervisor
Known by
Major of the Livestock Production
and Technology
Chainnan,
Final defense: 19 June 2014
Passed:
2 3 JUL 20'&
Examiners on preliminary defense:
1. Dr agr Asep Gunawan, SPt, MSc
2. Dr Ni Wayan Kurniani Karja, DVM, MP
Examiners on final defense:
1. Dr Ir Bess Tiesnamurti, MSc
2. Prof Dr M. Agus Setiadi, DVM
ACKNOWLEDGEMENT
Many thanks to supervising committee, Prof Dr Ir Cece Sumantri, MAgrSc,
Prof Arief Boediono, DVM, PhD, PAVet (K), Prof Dr Ir Ronny R. Noor, MRurSc
and Prof Dr Göran Andersson, who has provided guidance and assistance during
my study at the Graduate School, Bogor Agricultural University (IPB), Bogor,
Indonesia and Department of Animal Breeding and Genetics, Swedish University
of Agricultural Sciences (SLU), Uppsala, Sweden.
Sincere appreciation presented to the main sponsor, Graduate Scholarship
Program from Ministry for Research and Technology (2010-2014) and the Guest
Scholarship Program from Swedish Institute (2011-2013), which has provided
financial support for the implementation of study and research. My appreciation
also presented to Research Centre of Biotechnology, Indonesian Institute of
Sciences.
My high appreciation is also to Dr Ni Wayan Kurniani Karja, DVM, MP, Dr
agr Asep Gunawan, SPt, MSc, Dr Ir Bess Tiesnamurti, M.Sc and Prof Dr
Mohamad Agus Setiadi, DVM as examiners, which have provided feedback and
comments that are very useful for improving this dissertation. To the Head of
Animal Production and Technology study program at the Graduate School, IPB,
the Head of Department of Animal Breeding and Genetic, Swedish University of
Agricultural Science (SLU) and his staff, Prof Dr Leif Andersson, all my teachers,
technical assistance and administrative staff, I presented infinite gratitude for all
help and support that given to me during my study.
Special thanks and appreciation for help and cooperation to all collaborators
and co-authors, Dr Muhammad Rizal, Dr Hera Maheshwari, Dr Maria Wilbe and
Astri Olivia Herlino, MSc. Many thanks to Ir Isak Maraya Allosomba, MSi, Ir
Andarias Tandung Sale, MSi, the Governmental Animal Division of North Toraja
District, buffalo owners and farmers, who have given full support for collecting
samples and performing this study. To Dr Ir Niken Ulupi, MSi and Dr Ir M.
Amrullah Pagala, MSi, thank you very much for friendship, support and help.
Special thanks to Dr Ir Takdir Saili, MSi, Prof Dr Herdis, DVM, MSi, Dr Ir
Ristika Handarini, MP, Kemaz Aditya Dewangga, DVM, LL B, MPN and Agus
Teguh Suryaman, BVM, LL B, LL M, who had come and given support during
the dissertation defense.
My parents, brother, sister and families, thank you for love and supports that
given to me. Mama, Papa and Claudi, thank you for support and love. Last but not
least, special thanks to Tobias Wellnitz, for love, support, help and great
comments in my dissertation.
Bogor, June 2014
Yulnawati
i
TABLE OF CONTENTS
TABLE LISTS
ii
FIGURE LISTS
iii
1
2
INTRODUCTION
Background
General Objectives
Hypothesis
Outputs
Novelties
1
3
3
3
4
SPOTTED COAT COLOR PATERN AND ITS EFFECT ON THE
QUALITY OF EPIDIDYMAL SPERM
Introduction
Materials and Methods
Results and Discussion
Conclusion
7
10
12
14
THE QUALITY AND FERTILITY OF FROZEN-THAWED
EPIDIDYMAL SPERM IN TWO DIFFERENT EXTENDERS
Introduction
Materials and Methods
Results and Discussion
Conclusion
15
16
17
20
MITF MUTATIONS ARE CAUSING SPOTTED COAT COLOR IN
SWAMP BUFFALOES
Introduction
Materials and Methods
Results and Discussion
Conclusion
21
25
27
33
5
GENERAL DISCUSSION
35
6
CONCLUSION AND RECOMMENDATION
39
3
4
REFERENCES
41
SUPPLEMENTS
Sequence Listing of MITF Gene
49
55
BIOGRAPHY
61
ii
TABLE LISTS
2.1
2.2
2.3
3.1
3.2
3.3
4.1
4.2
S.1
Classification of swamp buffalo based on coat pattern and iris
color
The quality of fresh epididymal sperm
The quality of frozen-thawed epididymal sperm
The composition of sperm extenders
The quality of fresh epididymal sperm of spotted buffalo
The quality of epididymal sperm during cryopreservation and
after thawing
Identified SNPs in swamp buffalo MITF gene
The genotype frequencies of the nonsense mutation (chr22:
32,322,242a) and the splice site mutation (chr22: 32,297,683a) in
MITF gene of swamp buffaloes with different coat color
phenotypes
The accession numbers of identified SNPs in MITF gene
8
13
14
16
18
19
27
29
54
iii
FIGURE LISTS
1.1
1.2
2.1
2.2
2.3
4.1
4.2
4.3
4.4
4.5
4.6
4.7
4.8
5.1
S.1
S.2
S.3
S.4
The estimated spotted buffalo population over the last five years
in Northern Toraja District
The framework of studies
A compilation data of buffalo classification and domestication
time period
Different type of coat color pattern in swamp buffalo
Testicle and epididymis of buffalo bull
The schema of MITF gene that uses cattle genome as reference
The roles of action of MITF in pigmentation
The results of Sanger sequencing of MITF DNA products
generated from mutant spotted and wild-type swamp buffaloes
Variations of coat and eye color phenotype in swamp buffaloes
The mRNA sequences of wild-type versus splice-site mutation
Amino acids alignment of splice-site mutation (SS) of spotted
buffaloes versus wild-type of solid buffaloes (Wt) and 9 other
species
The binding affinity of mutant versus wild-type MITF protein on
electrophoretic mobility shift assay (EMSA)
The estimated position of splice-site mutation in MITF gene and
the position of helix-loop-helix DNA binding (HLH DNA-bd)
and the leucine zipper part in the basic helix-loop-helix zipper
(bHLH-Zip) domain
The estimation of the offspring (F1) coat color based on
Mendelian formulation in swamp buffalo
Offspring generated from AI using frozen-thawed epididymal
sperm of a Saleko buffalo bull
Eighteen months old heifers, generated from AI using frozenthawed epididymal sperm of Saleko male and solid female
buffaloes
A Saleko spotted buffalo bull as the source of epididymal sperm
that were used for AI in our study
The mating pattern of swamp buffalo with different coat color
phenotype and the coat color of the offspring
1
5
8
9
10
23
24
28
29
30
30
31
32
36
51
52
52
53
1
INTRODUCTION
Background
The swamp buffalo (lat. Bubalus bubalis carabanensis) is an important
domestic animal in Indonesia. Particularly in the Toraja region, South Sulawesi,
the role of swamp buffalo goes beyond its general significance in agriculture. For
the indigenous ethnic group Toraja, swamp buffalo bulls with a spotted coat color
pattern and white iris color are considered as holy animals. A fundamental part of
Toraja’s animalistic beliefs is the sacrificial offering of these holy animals in large
funeral ceremonies, called Rambu Solo’. During those ceremonies, dozens of
animals, mainly spotted buffalo bulls but also cattle, pigs, horses, deer, and
chickens, are sacrificed as an expression of sorrow and condolence. According to
their spiritual beliefs, spotted buffalo bulls will give the deceased person a safe
ride to heaven. Despite that most of Toraja’s population has converted to
Christianity (>90%) or Islam (
CAUSATIVE MUTATIONS RELATED TO SPOTTED
COAT COLOR PATTERN IN SWAMP BUFFALO
YULNAWATI
GRADUATE SCHOOL
BOGOR AGRICULTURAL UNIVERSITY
BOGOR
2014
STATEMENT OF COPYRIGHT FOR DISSERTATION AND
SOURCES OF INFORMATION *
I hereby declare that the dissertation entitled “The Utilization of
Epididymal Sperm and the Causative Mutations Related to Spotted Coat
Color Pattern in Swamp Buffalo” is true of my work under the guidance of the
supervising committee and has not been submitted in any form to any college.
Resources derived or quoted from works published and unpublished from other
writers mentioned in the text and listed in the References at the end of this
dissertation.
I hereby bestow the copyright of my papers to the Bogor Agricultural
University (IPB).
Bogor, June 2014
Yulnawati
NIM D161100041
RINGKASAN
YULNAWATI. Pemanfaatan Sperma Epididimis dan Mutasi Penyebab
Munculnya Warna Belang Pada Kerbau Lumpur. Dibimbing oleh CECE
SUMANTRI, ARIEF BOEDIONO, RONNY RACHMAN NOOR dan GÖRAN
ANDERSSON.
Kerbau belang merupakan salah satu keanekaragaman hayati Indonesia
yang termasuk dalam kelompok kerbau lumpur atau kerbau rawa (Bubalus
bubalis carabanensis). Populasi kerbau belang umumnya ditemui di sekitar
wilayah Toraja, Sulawesi Selatan. Jenis ternak ini memiliki ikatan yang sangat
erat dengan kebudayaan setempat. Kerbau belang jantan dikorbankan untuk
dijadikan sebagai persembahan pada saat upacara kematian. Rendahnya tingkat
kelahiran akibat tradisi yang tidak menginginkan terjadinya kawin alam pada
kerbau belang jantan, serta tingkat pemotongan yang tinggi akibat upacara adat,
menyebabkan populasi ternak ini menurun drastis, mendekati kepunahan.
Disertasi ini bertujuan untuk i) menginvestigasi metode reproduksi yang
dapat diterapkan untuk meningkatkan angka kelahiran kerbau belang, dan ii)
mengidentifikasi mutasi pada gen MITF sebagai penyebab munculnya variasi
warna belang. Disertasi ini terdiri dari tiga kajian yang meliputi aspek reproduksi
kerbau belang jantan dan variasi genetik penyebab munculnya warna belang pada
kerbau rawa.
Pada kajian pertama, dilakukan investigasi mengenai pengaruh warna kulit
terhadap kualitas sperma epididimis segar dan setelah thawing. Sampel sperma
epididimis dikoleksi dari 12 ekor kerbau belang yang terdiri dari 5 ekor Saleko, 4
ekor Bonga dan 3 ekor Lotong Boko, dibandingkan dengan sperma epididimis dari
5 ekor kerbau hitam. Tidak ditemukan adanya perbedaan signifikan pada setiap
parameter kualitas sperma yang diamati, baik pada kondisi sperma segar maupun
setelah thawing. Motilitas progresif sperma epididimis setelah thawing dari
kelompok Saleko, Bonga, Lotong Boko dan hitam secara berturut-turut adalah
44%, 42%, 40% dan 42%. Sementara itu, daya hidup dan keutuhan membran
plasma sperma epididimis dari keempat kelompok tersebut secara berturut-turut
adalah 64,9%; 65,2%; 62,6%; 62,7% and 64,6%; 67,1%; 64,5%; 64,1%.
Disimpulkan bahwa variasi warna kulit belang ternyata tidak memberikan
pengaruh terhadap kualitas sperma epididimis segar maupun beku.
Kajian kedua bertujuan untuk mengetahui pengaruh dua bahan pengencer
terhadap kualitas sperma epididimis setelah thawing, serta keberhasilan
kebuntingan setelah inseminasi buatan (IB) menggunakan sperma tersebut. Bahan
pengencer yang dibandingkan adalah Tris kuning telur (TEY20) versus Sitrat
kuning telur (CEY20). Hasil yang diperoleh menunjukkan kedua jenis pengencer
memiliki kemampuan yang sama dalam mempertahankan motilitas progresif,
viabilitas dan keutuhan membran plasma sperma epididimis setelah thawing.
Sebanyak 47% (7/15) kerbau betina berhasil bunting setelah diinseminasi
menggunakan sperma epididimis setelah thawing yang diencerkan dalam TEY20
dan 40% (6/15) menggunakan sperma epididimis setelah thawing yang diencerkan
dalam CEY20. Disimpulkan bahwa kedua jenis bahan pengencer, TEY20 dan
CEY20, dapat digunakan untuk mempertahankan kualitas dan fertilitas sperma
epididimis kerbau belang setelah thawing.
Dalam kajian ketiga, dilakukan penelusuran variasi genetik pada gen
microphthalmia-associated transcription factor (MITF) dalam kaitannya terhadap
kemunculan warna belang. Studi ini berhasil mengidentifikasi dua jenis mutasi
penting, yaitu mutasi nonsense yang menyebabkan terjadinya premature stop
codon pada exon 3, dan mutasi donor splice-site yang menyebabkan perpanjangan
exon 8. Akibat mutasi splice-site ini terjadinya penambahan 8 residu asam amino
yang diperkirakan terjadi langsung sebelum leucine ketiga dari leucine zipper
bHLH-Zip domain, sehingga mempengaruhi dimerisasi dan kapasitas ikatan
protein MITF dengan DNA.
Secara umum dapat disimpulkan bahwa IB menggunakan sperma epididimis
kerbau belang yang dibekukan baik dalam bahan pengencer TEY20 maupun
CEY20 dapat diaplikasikan guna meningkatkan angka kelahiran kerbau belang.
Selanjutnya, dapat dipastikan bahwa mutasi nonsense dan splice-site yang
diidentifikasi pada gen MITF merupakan faktor penyebab munculnya warna
belang pada kerbau rawa.
Kata kunci: Sperma Epididimis, Bahan Pengencer, Inseminasi Buatan, MITF,
Kerbau Belang
SUMMARY
YULNAWATI. The Utilization of Epididymal Sperm and Causative Mutations
Related to Spotted Coat Color Pattern in Swamp Buffalo. Supervised by CECE
SUMANTRI, ARIEF BOEDIONO, RONNY RACHMAN NOOR and GÖRAN
ANDERSSON.
The spotted buffalo is an Indonesian domestic buffalo, which is a variant of
the swamp buffalo (Bubalus bubalis carabanensis) type. The spotted buffalo is
mainly found in the Toraja region, South Sulawesi. Apart from its importance in
agriculture, this species is also closely tied to local spiritual beliefs. The sacrificial
offering of spotted buffalo is considered a fundamental part of funeral
ceremonies. Due to the spotted buffalo’s low birth rate of natural reproduction,
but yet high demand in funeral ceremonies, the species is close to extinction.
The objectives of this dissertation were i) to investigate the applicative
reproductive methods that are useful to increase the birth rate of spotted buffalo,
and ii) to identify the causative mutations and variations in MITF gene that are
associated with spotted coat color pattern in swamp buffalo. This dissertation
consists of three studies, two studies were related to the reproductive aspect of
spotted buffalo bulls and the third study was related to the variation of a
pigmentation gene in swamp buffalo.
In the first study we investigated if fresh and frozen-thawed epididymal
sperm quality was influenced by the buffalo’s coat color pattern. Therefore,
epididymal sperm from 12 buffaloes with spotted coat color pattern (5 Saleko, 4
Bonga and 3 Lotong Boko) were compared to sperm from 5 buffaloes with solid
coat color pattern. Results showed that the coat color pattern did not influence the
sperm quality. No significant difference was found in any of the analyzed
parameters among groups of fresh and frozen-thawed sperm. The percentage of
frozen-thawed progressive motility from Saleko, Bonga, Lotong Boko and Solid
was 44%, 42%, 40% and 42%, respectively. Moreover, the percentage of viability
and membrane integrity of frozen-thawed sperm were 64.9%; 65.2%; 62.6%;
62.7% and 64.6%; 67.1%; 64.5%; 64.1%, respectively. In conclusion, the buffalo
coat color pattern variation did not affect the quality of fresh and frozen-thawed
epididymal sperm.
The objective of the second study was to investigate the ability of
chemically defined extenders in maintaining the quality and fertility of frozenthawed epididymal sperm of spotted buffaloes. Two extenders, Tris-based egg
yolk (TEY20) and Citrate-based egg yolk (CEY20), were used in this study. The
results showed a similar progressive motility, viability and membrane integrity of
frozen-thawed epididymal sperm in both extenders. With 30 buffalo cows, the
resulting success rate of pregnancy was 47% (7/15) using frozen-thawed
epididymal sperm in TEY20 and 40% (6/15) using the one in CEY20 extender. In
conclusion, the two chemically defined extenders (TEY20 and CEY20) were
equally suitable to maintain the quality and fertility of frozen-thawed epididymal
sperm.
In the third study we identified the genetic variation in the microphthalmia
associated transcription factor (MITF) gene that is associated to the spotted coat
color pattern. We identified two important causative mutations: first, a nonsense
mutation that caused premature stop codon in exon 3 and likely removal of the
resulting mRNA via nonsense-mediated mRNA decay pathway; second, a donor
splice-site mutation that led to aberrant mRNA splicing of exon 8 of MITF
resulting in an mRNA coding for protein with eight additional amino acid
residues. Most likely the insertion of the eight amino acid residues negatively
influences dimerization and DNA binding capacity because the insertion was
located immediately before the third leucine in the leucine zipper bHLH-Zip
domain.
In conclusion, AI using frozen-thawed epididymal sperm, which was diluted
either in TEY20 or CEY20, could be applied to increase the birth rate of spotted
buffaloes. Furthermore, the nonsense and splice-site mutations in MITF gene were
strongly associated with spotted coat color pattern in swamp buffaloes.
Keywords: Epididymal Sperm, Extenders, Artificial Insemination, MITF, Spotted
Buffalo
© All Rights Reserved IPB, in 2014
Copyright Reserved
Prohibited from quoting part or all of this paper without including or citing
sources. Citations only for educational purposes, research, writing papers,
preparing reports, writing criticism, or review a matter, and citations are not
detrimental to the interests of the University.
Prohibited announced and reproduce part or all of this publication in any form
without permission from IPB.
THE UTILIZATION OF EPIDIDYMAL SPERM AND
CAUSATIVE MUTATIONS RELATED TO SPOTTED
COAT COLOR PATTERN IN SWAMP BUFFALO
YULNAWATI
Dissertation
As a requirements for Doctoral degree
at
Major of Livestock Production and Technology
GRADUATE SCHOOL
BOGOR AGRICULTURAL UNIVERSITY
BOGOR
2014
Title
Name
Student ID
: The Utilization ofEpidiymal Spenn and Causative Mutations
Related to Spotted Coat Color Pattern in Swamp Buffalo
: Yulnawati
: D161100041
Approved by
Supervision Committee
Prof Dr Ir Cece Sumantri, MAgrSc Prof AriefBoediono, DVM, PhD, PAVet (K)
Main Supervisor
Co-Supervisor
Prof Dr Ir Ronny R. Noor, MRurSc
Co-Supervisor
Prof Dr Goran Andersson
Co-Supervisor
Known by
Major of the Livestock Production
and Technology
Chainnan,
Final defense: 19 June 2014
Passed:
2 3 JUL 20'&
Examiners on preliminary defense:
1. Dr agr Asep Gunawan, SPt, MSc
2. Dr Ni Wayan Kurniani Karja, DVM, MP
Examiners on final defense:
1. Dr Ir Bess Tiesnamurti, MSc
2. Prof Dr M. Agus Setiadi, DVM
ACKNOWLEDGEMENT
Many thanks to supervising committee, Prof Dr Ir Cece Sumantri, MAgrSc,
Prof Arief Boediono, DVM, PhD, PAVet (K), Prof Dr Ir Ronny R. Noor, MRurSc
and Prof Dr Göran Andersson, who has provided guidance and assistance during
my study at the Graduate School, Bogor Agricultural University (IPB), Bogor,
Indonesia and Department of Animal Breeding and Genetics, Swedish University
of Agricultural Sciences (SLU), Uppsala, Sweden.
Sincere appreciation presented to the main sponsor, Graduate Scholarship
Program from Ministry for Research and Technology (2010-2014) and the Guest
Scholarship Program from Swedish Institute (2011-2013), which has provided
financial support for the implementation of study and research. My appreciation
also presented to Research Centre of Biotechnology, Indonesian Institute of
Sciences.
My high appreciation is also to Dr Ni Wayan Kurniani Karja, DVM, MP, Dr
agr Asep Gunawan, SPt, MSc, Dr Ir Bess Tiesnamurti, M.Sc and Prof Dr
Mohamad Agus Setiadi, DVM as examiners, which have provided feedback and
comments that are very useful for improving this dissertation. To the Head of
Animal Production and Technology study program at the Graduate School, IPB,
the Head of Department of Animal Breeding and Genetic, Swedish University of
Agricultural Science (SLU) and his staff, Prof Dr Leif Andersson, all my teachers,
technical assistance and administrative staff, I presented infinite gratitude for all
help and support that given to me during my study.
Special thanks and appreciation for help and cooperation to all collaborators
and co-authors, Dr Muhammad Rizal, Dr Hera Maheshwari, Dr Maria Wilbe and
Astri Olivia Herlino, MSc. Many thanks to Ir Isak Maraya Allosomba, MSi, Ir
Andarias Tandung Sale, MSi, the Governmental Animal Division of North Toraja
District, buffalo owners and farmers, who have given full support for collecting
samples and performing this study. To Dr Ir Niken Ulupi, MSi and Dr Ir M.
Amrullah Pagala, MSi, thank you very much for friendship, support and help.
Special thanks to Dr Ir Takdir Saili, MSi, Prof Dr Herdis, DVM, MSi, Dr Ir
Ristika Handarini, MP, Kemaz Aditya Dewangga, DVM, LL B, MPN and Agus
Teguh Suryaman, BVM, LL B, LL M, who had come and given support during
the dissertation defense.
My parents, brother, sister and families, thank you for love and supports that
given to me. Mama, Papa and Claudi, thank you for support and love. Last but not
least, special thanks to Tobias Wellnitz, for love, support, help and great
comments in my dissertation.
Bogor, June 2014
Yulnawati
i
TABLE OF CONTENTS
TABLE LISTS
ii
FIGURE LISTS
iii
1
2
INTRODUCTION
Background
General Objectives
Hypothesis
Outputs
Novelties
1
3
3
3
4
SPOTTED COAT COLOR PATERN AND ITS EFFECT ON THE
QUALITY OF EPIDIDYMAL SPERM
Introduction
Materials and Methods
Results and Discussion
Conclusion
7
10
12
14
THE QUALITY AND FERTILITY OF FROZEN-THAWED
EPIDIDYMAL SPERM IN TWO DIFFERENT EXTENDERS
Introduction
Materials and Methods
Results and Discussion
Conclusion
15
16
17
20
MITF MUTATIONS ARE CAUSING SPOTTED COAT COLOR IN
SWAMP BUFFALOES
Introduction
Materials and Methods
Results and Discussion
Conclusion
21
25
27
33
5
GENERAL DISCUSSION
35
6
CONCLUSION AND RECOMMENDATION
39
3
4
REFERENCES
41
SUPPLEMENTS
Sequence Listing of MITF Gene
49
55
BIOGRAPHY
61
ii
TABLE LISTS
2.1
2.2
2.3
3.1
3.2
3.3
4.1
4.2
S.1
Classification of swamp buffalo based on coat pattern and iris
color
The quality of fresh epididymal sperm
The quality of frozen-thawed epididymal sperm
The composition of sperm extenders
The quality of fresh epididymal sperm of spotted buffalo
The quality of epididymal sperm during cryopreservation and
after thawing
Identified SNPs in swamp buffalo MITF gene
The genotype frequencies of the nonsense mutation (chr22:
32,322,242a) and the splice site mutation (chr22: 32,297,683a) in
MITF gene of swamp buffaloes with different coat color
phenotypes
The accession numbers of identified SNPs in MITF gene
8
13
14
16
18
19
27
29
54
iii
FIGURE LISTS
1.1
1.2
2.1
2.2
2.3
4.1
4.2
4.3
4.4
4.5
4.6
4.7
4.8
5.1
S.1
S.2
S.3
S.4
The estimated spotted buffalo population over the last five years
in Northern Toraja District
The framework of studies
A compilation data of buffalo classification and domestication
time period
Different type of coat color pattern in swamp buffalo
Testicle and epididymis of buffalo bull
The schema of MITF gene that uses cattle genome as reference
The roles of action of MITF in pigmentation
The results of Sanger sequencing of MITF DNA products
generated from mutant spotted and wild-type swamp buffaloes
Variations of coat and eye color phenotype in swamp buffaloes
The mRNA sequences of wild-type versus splice-site mutation
Amino acids alignment of splice-site mutation (SS) of spotted
buffaloes versus wild-type of solid buffaloes (Wt) and 9 other
species
The binding affinity of mutant versus wild-type MITF protein on
electrophoretic mobility shift assay (EMSA)
The estimated position of splice-site mutation in MITF gene and
the position of helix-loop-helix DNA binding (HLH DNA-bd)
and the leucine zipper part in the basic helix-loop-helix zipper
(bHLH-Zip) domain
The estimation of the offspring (F1) coat color based on
Mendelian formulation in swamp buffalo
Offspring generated from AI using frozen-thawed epididymal
sperm of a Saleko buffalo bull
Eighteen months old heifers, generated from AI using frozenthawed epididymal sperm of Saleko male and solid female
buffaloes
A Saleko spotted buffalo bull as the source of epididymal sperm
that were used for AI in our study
The mating pattern of swamp buffalo with different coat color
phenotype and the coat color of the offspring
1
5
8
9
10
23
24
28
29
30
30
31
32
36
51
52
52
53
1
INTRODUCTION
Background
The swamp buffalo (lat. Bubalus bubalis carabanensis) is an important
domestic animal in Indonesia. Particularly in the Toraja region, South Sulawesi,
the role of swamp buffalo goes beyond its general significance in agriculture. For
the indigenous ethnic group Toraja, swamp buffalo bulls with a spotted coat color
pattern and white iris color are considered as holy animals. A fundamental part of
Toraja’s animalistic beliefs is the sacrificial offering of these holy animals in large
funeral ceremonies, called Rambu Solo’. During those ceremonies, dozens of
animals, mainly spotted buffalo bulls but also cattle, pigs, horses, deer, and
chickens, are sacrificed as an expression of sorrow and condolence. According to
their spiritual beliefs, spotted buffalo bulls will give the deceased person a safe
ride to heaven. Despite that most of Toraja’s population has converted to
Christianity (>90%) or Islam (