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Lampiran 1 Prosedur pembuatan larutan pembilas testes
Larutan Pembilas (NaCl Fisiologis)
Bahan: 1. Serbuk NaCl
0,82 gr
2. Milli-Q
100 ml
Cara pembuatan : bahan dicampur merata dan disterilisasi dengan autoclave pada suhu 1500 C selama 15 menit.
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Lampiran 2 Komposisi Larutan Medium untuk Spermatofor dan Spermatozoa larutan Ca 2+ - free saline ( 1 L)
Komposisi
Jumlah (g)
NaCl
21.63
KCl
1.12
H3BO3
0.53
NaOH
0.19
MgSO4.7H2O
4.93
Catatan: pH 7.4 dengan 1 N HCl
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Lampiran 3
Prosedur SEM dari Spermatozoa Kepiting Bakau Merah (Scylla olivacea)
a. Preparasi SEM b. Buat kotak kecil dari objek glass (tetes dengan neofren 2,5%) c. Teteskan spermatozoa (setelah disentrifugasi untuk menghilangkan pengencer) d. Perendaman dengan glutaradehida 2,5 % (sebagai fiksatif) selama 1-2 jam e. Washing dengan PBS ( 3 kali @ 5 menit) f. Rendam dengan asam tannin 2 % in DW selama 1jam g. Washing dengan PBS sampai jernih h. Osmium tetraoxyde O5O4 1 % in DW selama 1 jam (cukup ditetes) i. Washing dengan PBS j. Dehidrasi dengan alkohol 70%-100% @ 1 jam (alkohol 100 %, 3 kali) k. Dehidrasi II dengan t-butanol (propylene oxyde atau aseton pa, 3 kali) l. Sediaan dikeringkan menggunakan freezedryer m. Tempelkan dilogam n. Coating dengan platinum o. Sediaan diamati dengan SEM
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Lampiran 4 Uji Statistika dengan SPSS Data Konsentrasi Spermatozoa per Spermatofor Scylla olivacea
UJI ANOVA
Konsentrasi Spermatozoa per Spermatofor Sum of Squares 225.870 95.409 321.279
Between Groups Within Groups Total
df
Mean Square 56.468 9.541
4 10 14
F 5.919
Sig. .010
Duncan Subset for alpha = .05 perlakuan 40-70
N 3
1 6.6700
70-100
3
10.1200
130-160
3
15.8767
15.8767
100-130
3
15.9633
15.9633
160-190
3
Sig.
2
3
10.1200
16.1433 .201
.051
.922
Means for groups in homogeneous subsets are displayed. a Uses Harmonic Mean Sample Size = 3.000.
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