Pengaruh larutan disosiasi dan lama masa inkubasi terhadap jumlah sel
Uji lanjut beda nyata faktor lama waktu inkubasi terhadap viabilitas sel
Post Hoc test
Viabilitas
lama inkubasi jam
Subset N
1 2
3 Duncan
a,,b
5 6
92,9833 4
6 95,6033
95,6033 3
6 97,4183
97,4183 2
6 99,0733
1 6
100,0000 Sig.
0,078 0,213
0,098 Means for groups in homogeneous subsets are displayed.
Based on observed means. The error term is Mean SquareError = 5.981.
a. Uses Harmonic Mean Sample Size = 6.000. b. Alpha = 0,05.
119
Lampiran 4 Profil resipien larva ikan nila pada berbagai umur hari pascamenetas hpm. Tanda panah menunjukkan daerah yang disuntik. Skala=1
mm
Lampiran 5 Persentase sel testikular dan sel spermatogonia ikan gurami yang terlabel oleh PKH 26 Sigma
Lapang Pandang Ikan donor pertama
Ikan donor kedua 1
2 3
1 2
3 Sel testikular
20 18
15 28
23 18
Sel terlabel 18
17 14
25 22
16 persentase sel terlabel
90,00 94,44 93,33 89,29 95,65 88,89 Rerata sel terlabel
91,93±2,90 Spermatogonia
3 2
2 2
2 4
Sel terlabel 2
1 2
1 2
2 Persentase sel terlabel
66,67 50,00 100,00 50,00 100,00 50,00 Rerata sel terlabel
69,44±24,53
120
Lampiran 6 Sintasan ikan nila pascatransplantasi
Perlakuan hari pascamenetas
Ulangan Jumlah ikan
ditransplantasi Sintasan pascatransplantasi
24 jam 2 bulan
1 1
20 75,00
20,00 2
40 87,50
77,50 3
35 85,71
65,71 Rerata
82,74±6,76 54,40±30,37
3 1
25 100,00
64,00 2
40 90,00
55,00 3
40 95,00
70,00 Rerata
95,00±5,00 63,00±7,55
5 1
20 95,00
60,00 2
40 92,50
75,00 3
40 97,50
90,00 Rerata
95,00±2,50 82,96±15,00
7 1
30 100,00
83,33 2
40 90,00
80,00 3
40 92,50
82,50 Rerata
94,17±5,20 81,94±1,73
Kontrol 1
20 100,00
75,00 2
40 100,00
80,00 3
35 100,00
80,00 Rerata
100,00±0,00 82,50±2,50
UJI ANOVA UNTUK SINTASAN LARVA PASCATRANSPLANTASI
Sum of Squares df Mean Square F
Sig. 24 jam
Between Groups 488,827 4
122,207 5,873
0,011 Within Groups
208,078 10
20,808 Total
696,905 14
2 bulan Between Groups 1376,710
4 344,178
1,396 0,304
Within Groups 2646,890
10 246,489
Total 3841,601
14
121
24 jam
Umur resipien
Subset for alpha = 0.05 N
1 2
Duncan
a,,b
1 3
82,7381 7
3 94,1667
3 3
95,0000 5
3 95,0000
4 100,0000
Sig. 1,000
0,175
2 bulan
Umur resipien
Subset for alpha = 0.05 N
1 Duncan
a,,b
1 3
54,4048 7
3 63,8889
3 3
75,0000 5
3 78,3333
4 79,1667
Sig. 0,106
122
Lampiran 7 Efisiensi kolonisasi sel spermatogonia ikan gurami pada resipien ikan nila
Perlakuan hari pascamenetas
Ulangan Jumlah sampel
Jumlah sampel yang membawa sel donor
Efisiensi kolonisasi
Jantan Betina
Jantan Betina
1
1 2
2 1
25 2
2 2
1 1
50 3
3 1
2 1
75 rerata
50±25
3
1 2
2 2
50 2
1 2
1 33,33
3 3
2 3
2 100
rerata 61,1±34,71
5
1 2
2 1
1 50
2 3
1 1
25 3
3 1
2 1
75 rerata
50±25
7
1 2
1 1
33,3 2
3 1
1 25
3 3
2 rerata
19,43±17,33
ANOVA EFISIENSI KOLONISASI
Sum of Squares df
Mean Square F
Sig. Between Groups
2887,796 3
962,599 1,398
0,312 Within Groups
5509,352 8
688,669 Total
8397,148 11
Lampiran 8 Hasil pengukuran ekstraksi DNA gonad ikan gurami menggunakan gene quant
Sampel Rasio 260280 Kandungan DNA µgµL
Keterangan 11
-1,381 tidak digunakan
12 1,659
0,032 pascapurifikasi
13 1,7
0,240 pascapurifikasi
14 1,679
0,048 pascapurifikasi
15 1,778
0,040 pascapurifikasi
16 0,778
tidak digunakan 21
1,815 0,152
22 1,783
0,416 23
1,756 0,056
24 2,291
0,016 25
1,775 0,192
26 1,652
0,072 31
1,685 1,296
32 1,83
3,576 33
1,702 1,020
34 1,761
0,080 35
1,756 3,284
36 1,845
3,112 L1
1,744 0,596
L2 1,761
0,712 L3
1,837 0,688
ST1 1,871
0,288 ST2
1,802 0,168
ST3 1,882
0,128 ST4
1,750 0,088
ST5 1,727
0,068 G
1,745 0,144
N 1,849
0,064 Keterangan
11-16 = Gonad resipien ikan nila 1 bulan pt
21-26 = Gonad resipien ikan nila 2 bulan pt 31-36 = Gonad resipien ikan nila 3 bulan pt
L1-L3 = Resipien larva ikan nila 24 jam pt ST1
= Suspensi sel tesikular ikan gurami dan nila rasio 100000:400000 ST2
= Suspensi sel tesikular ikan gurami dan nila rasio 10000:490000 ST3
= Suspensi sel tesikular ikan gurami dan nila rasio 1000:499000 ST4
= Suspensi sel tesikular ikan gurami dan nila rasio 100:499900 ST5
= Suspensi sel tesikular ikan gurami dan nila rasio 10:499990 G
= Suspensi sel testikular ikan gurami N
= Gonad ikan nila tanpa transplantasi
Lampiran 9 Hasil kuantifikasi DNA produk PCR DNA genom gonad ikan nila menggunakan program unscan IT Gel 6.1
1. Elektroforegram DNA produk PCR DNA genom larutan standar SD dan larva 24 jam pascatransplantasi
SegmenSampel Pixel Total
Concentration Pixel Total
Segment Size
Segment Size
1.Marker 500 bp 57.198
14,28 6,96
456 7,2
2. Marker 1000 bp 57.386
14,53 6,98
462 7,3
3.24 jam 80.503
45,35466 9,8
660 10,42
5.24 jam 136.743
120,3454 16,64
782 12,35
6.24 jam 69.534
30,72854 8,46
630 9,95
7.G 107.050
80,75256 13,03
552 8,72
8.100.000 162.553
154,7605 19,78
752 11,88
9.10.000 52.548
8,079326 6,4
598 9,44
10.1.000 49.447
3,944437 6,02
720 11,37
11.100 48.718
2,972385 5,93
720 11,37
konsentrasi DNA standar
2. Elektroforegram DNA produk PCR DNA genom gonad ikan nila umur 1 bulan pascatransplantasi.
SegmenSampel Pixel Total
Concentration Pixel
Total Segment
Size Segment
Size 1.Marker 500 bp
124.215 14,28
20,27 960
14,38 2.Marker 1000 bp
118.440 14,53
19,32 1.280
19,17 3.12
169.799 12,30642
27,7 1.771
26,52 4.13
200.492 10,97756
32,71 2.666
39,93
konsentrasi DNA standar
3. Elektroforegram DNA produk PCR DNA genom gonad ikan nila umur 2 bulan dan 3 bulan pascatransplantasi
SegmenSampel Pixel Total
Concentration Pixel
Total Segment
Size Segment
Size 1.Marker 1000 bp
290.640 14,28
4,45 2.496
5,42 2.Marker 500 bp
299.723 14,53
4,59 2.604
5,66 3.31
395.818 17,1739
6,07 3.710
8,06 4.32
419.128 17,81525
6,42 4.214
9,15 4.34
274.798 13,84417
4,21 3.456
7,51 5.35
382.579 16,80964
5,86 3.520
7,65 6.36
878.062 30,4423
13,46 4.992
10,85 7.G
513.354 20,40777
7,87 3.094
6,72 8.21
806.373 28,46985
12,36 3.744
8,13 9.22
766.415 27,37046
11,74 3.488
7,58 10.23
613.787 23,17107
9,41 3.952
8,59 11.24
417.854 17,7802
6,4 2.600
5,65 12.25
301.396 14,57598
4,62 2.392
5,2 13.26
165.639 10,84078
2,54 1.768
3,84
konsentrasi DNA standar
Lampiran 10 Hasil analisis ragam estimasi konsentrasi dan jumlah sel resipien ikan nila 1 bulan, 2 bulan dan 3 bulan pascatransplantasi
Gonad dari resipien pasca transplantasi
Konsentrasi DNA ngµ L Jumlah sel
24 jam 25,92
29.583 24 jam
68,77 77.963
24 jam 17,56
20.147 1 bulan
0,00 323
1 bulan 3,52
4.293 1 bulan
3,14 3.864
1 bulan 0,00
323 2 bulan
16,27 18.690
2 bulan 15,64
17.981 2 bulan
13,24 15.272
2 bulan 10,16
11.794 2 bulan
8,33 9.727
2 bulan 6,19
7.317 3 bulan
9,81 11.403
3 bulan 10,18
11.816 3 bulan
0,00 323
3 bulan 7,91
9.254 3 bulan
9,61 11.168
3 bulan 17,40
19.963
ANOVA KONSENTRASI DNA DAN JUMLAH SEL IKAN GURAMI
Sum of Squares df Mean Square F
Sig. Konsentrasi
Between Groups 247,189 2
123,595 6,452 0,011
Within Groups 249,038
13 19,157
Total 496,227
15 Jumlah sel
Between Groups 3,152E8 2
1,576E8 6,459 0,011
Within Groups 3,172E8
13 2,440E7
Total 6,325E8
15
Uji beda nyata terhadap konsentrasi DNA
Duncan
a,,b
Perlakuan Subset for alpha = 0,05
N 1
2 1
4 1,6650
3 6
9,1517 2
6 11,6383
Sig. 1,000
0,379
Uji beda nyata terhadap jumlah sel
Duncan
a,,b
Perlakuan Subset for alpha = 0,05
N 1
2 1
4 2200,75
3 6
10654,50 2
6 13463,50
Sig. 1,000
0,378
Lampiran 11 Jumlah dan viabilitas spermatogonia ikan gurami dari testes pascapreservasi serta analisis ragam terhadap viabilitas
spermatogonia
Lama preservasi jam
Ulangan Jumlah
spermatogoniamg testes Viabilas
spermatogonia 1
27.556 96.77
2 25.333
100.00 3
41.333 93.55
6 1
44.211 84.13
2 40.580
91.43 3
44.667 89.55
12 1
28.406 75.51
2 30.725
81.13 3
32.381 76.47
24 1
10.526 80.00
2 14.902
73.68 3
8.667 69.23
48 1
26.133 44.90
2 5.797
60.00 3
27.333 58.54
ANOVA
Sum of Squares
df Mean Square F
Sig. viabilitas Between Groups
3079,162 4
769,790 29,058 0,000
Within Groups 264,918
10 26,492
Total 3344,080
14
Uji beda nyata
Duncan
a
Periode preservasi jam
Subset for alpha = 0,05 N
1 2
3 48
3 54,4800
24 3
74,3033 12
3 77,7033
6 3
88,3700 3
96,7733 Sig.
1,000 0,437
0,073
Lampiran 12 Sintasan resipien ikan nila 24 jam pascatransplantasi dan efisiensi kolonisasi resipien dengan sumber donor sel testikular ikan gurami
dari gonad pascapreservasi
Periode preservasi jam
Ulangan Sintasan 24 jam pt
Efisiensi kolonisasi 1
90 83,33
50 50
2 100
3 90
Rata-rata 93,33 ± 5,77
61,11 ± 19,24 24
1 90
85 100
66,67 50
50 2
3 Rata-rata
91,67 ± 7,64 55,56 ± 9,62
48 1
80 90
90 50
50 66,67
2 3
Rata-rata 86,67 ± 5,77
55,56 ± 9,62
ANOVA
Sum of Squares df
Mean Square Sintasan 24jam pt
Between Groups 72,222
2 36,111
Within Groups 250,000
6 41,667
Total 322,222
8 Sintasan 1 bulan pt Between Groups
16,667 2
8,333 Within Groups
883,333 6
147,222 Total
900,000 8
Efisiensi kolonisasi Between Groups 61,679
2 30,840
Within Groups 1111,111
6 185,185
Total 1172,790
8 F
Sig. Sintasan 24jam pt
Between Groups 0,867
0,467 Sintasan 1 bulan pt Between Groups
0,057 0,945
Efisiensi kolonisasi Between Groups 0,167
0,850
ANOVA SINTASAN DAN EFISIENSI KOLONISASI
Sum of Squares df Mean Square
F Sig.
Sintasan 24jam
Between Groups 72,222
2 36,111
0,0867 0,467 Within Groups
250,000 6
41,667 Total
322,222 8
Efisiensi kolonisasi
Between Groups 61,679
2 30,840
0,167 0,850
Within Groups 1111,111
6 185,185
Total 1172,790
8
DAFTAR ISTILAH MENURUT KAMUS
Derivat : turunan.
Diferensiasi : suatu tahap perkembangan embrio dari fase muda ke fase yang
definitif. Disosiasi
: pemisahan atau penguraian. Donor
: pemberi, masuk dan berintegrasi dengan inang Duktus deferens
: vas deferens, saluran mani yang menyalurkan mani dari epididimis ke urethra.
Epididymis : epididimis, anak testis pelir. Sperma dari vas efferensia
mengalami pematangan di epididimis sehingga jadi motil dan bergerak lalu disalurkan ke vas deferens.
Filogenetik : proses perkembangan evolusi makhluk hidup
Fluoresens : berbinar atau pendar fluor, membuat sediaan jadi berbinar jika
dilihat di bawah mikroskop fluorescence. Grid
: kisi; garis imajiner membujur dan melintang Immune
: imun,kebal Imunokompetensi
: mampu berespons imun Imunodefisiensi
; tak mampu menghasilkan imunitas. Larva
: suatu tahap perkembangan setelah sel telur menetas, memiliki bagian tubuh khas yang tidak dimiliki oleh tahap dewasa dan
aktif bergerak. Mutan
: sel atau individu yang terbentuk akibat mutasi . Nutfah
: benih Niche
: nike, relung yaitu cara hidup pada suatu habitat. Pluripoten
: memiliki potensi menumbuhkan atau berkembang menjadi beberapa macam sel.atau jaringan.
Preparat : bahan yang disiapkan secara kimiawi, sediaan
Preservasi : pengawetan, pemeliharaan.
Primordial germ cell PGC : calon atau bakal sel benih. Proliferasi
: penggandaan atau perbanyakan gametogonium secara mitosis untuk membentuk gametosit.
Resipien : penerima
Resiprok : berbalasan, sebaliknya. Perkawinan F1 betina x resesif jantan,
resiproknya F1 jantan x resesif betina. Testikular
: testis Tubuli seminiferi
: Jamak dari tubulus, saluran-saluran halus penghasil semen dalam testis.
Xenogeneic : xenogenik, berasal dari berbagai spesies.
Xenotransplantasi : Transplantasi xenogenik, transplantasi sel donor ke resipien
yang berbeda spesies.
iii
ABSTRACT
IRMA ANDRIANI. Xenotransplantation of Giant Gourami Testicular Germ Cells into Larvae of Nile Tilapia. Under direction of ALIMUDDIN, KOMAR
SUMANTADINATA, and ITA DJUWITA.
We were attempting to develop testicular germ cell TGC transplantation as a tool to produce surrogate broodstock of commercially
valuable fish and long generation time species. Giant gourami testis had been used as a model for donor and Nile tilapia larvae as recipient. The research was
conducted to study the competency of giant gourami TGC as donor and Nile tilapia larvae as recipient for xenotransplantation of giant gouramy TGC into
larvae of Nile tilapia. We developed TGC xenotransplantation system by some steps as follow : 1 The characterization of spermatogonia to identify optimal
donor of giant gourami based on body weight using histological approach, 2 The determination of dissociation method for giant gourami testicular tissue by
comparing two different composition of dissociation medium with 5 hours incubation time, 3 Optimizing the timing of intraperitoneal TGC transplantation
into peritoneal cavity of 1, 3, 5 and 7 days post hatching dph recipient by investigating the colonization efficiency of donor cell labelled PKH 26 fluorescent
membrane dye under fluorescent microscope and of molecular technique using growth hormone of giant gourami specific primer, 4 Analyzing the proliferation
of spermatogonia colonized in recipient gonad using molecular approach, and 5 The evaluation of TGC isolated from testis of giant gourami which preserved at
4
o
C using NaCl 0.7 for 6, 12, 24 and 48 hours and then transplanted into peritoneal cavity of recipient. The result showed that the donor with abundant
spermatogonia stem cell and A type spermatogonia cell diameter = 14.43 –20.53
µm were found in giant gourami with body weight ranged from 500 –1000 g. The
dissociation method produced high number of spermatogonia with high viability was one used medium PBS containing trypsin, DNase, CaCl
2
, HEPES, FBS and incubated for 3 hours. The highest colonization efficiency was observed at 3 dph
recipient 61.1±34.71 suggesting that 3 dph Nile tilapia larvae was the optimum recipient for transplantation. Intraperitoneally transplanted xenogenic
spermatogonia efficiently colonized the ovary as well with sex ratio male out of female was 1:1, and possibly proliferated indicated by cell cluster forming and the
increase of DNA concentration of donor in recipient testis during time interval 1 month to 2 or 3 month pt. The successful colonization of spermatogonia isolated
from preserved testis were also observed with colonization efficiency not differed significantly as from non preserved testis. In conclusion : the testis of giant
gouramy was composed of cells that had competency as donor for xenotransplantation using Nile tilapia larvae as recipient.
Key words : xenotransplantation, testicular germ cell, giant gourami, Nile tilapia, colonization efficiency, proliferation, preservation.