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Lampiran 1. Langkah-langkah Pengambilan Sampel

Langkah-langkah pengambilan sampel (Suhaemi, 2011):

1. Memberi nomor 1-30 pada tikus.

2. Membuat 30 gulungan kertas sesuai dengan jumlah kelompok perlakuan (KI, KII, KIII,

KIV, KV) dan jumlah ulangannya. Setiap kelompok perlakuan diulang sebanyak 6 kali.

Misal: KI.1 berarti kelompok perlakuan KI ulangan ke-1. KI.2 berarti kelompok

perlakuan KI ulangan ke-2, dan seterusnya sampai KI.6.

3. Membuat tabel dengan angka 1-30.
4. Melakukan pengocokan 30 gulungan kertas dan mengambil satu per satu. Kertas yang

keluar pertama dimasukkan pada tabel nomor 1. Misal: KII.5 keluar pertama diletakkan

pada tabel nomor 1 yang berarti tikus nomor 1 masuk kelompok perlakuan II ulangan 5.

1 2 3 4 5
5. Jika 6 7 8 9 10
11 12 13 14 15
16 17 18 19 20
21 22 23 24 25
26 27 28 29 30
pengambilan sampel secara random telah selesai, maka didapatkan 5 kelompok hewan

coba.

Lampiran 2. Komposisi Pakan Standar Comfeed

Tabel 3.3 Komposisi Pakan standar Comfeed

Zat gizi Kadar

Air Maksimal 12%
Protein kasar Minimal 15%
Lemak kasar 3-7%
 Serat kasar Maksimal 6%
Abu Maksimal 7%
Kalsium 0,9-1,1%
Fosfor 0,6-0,9%

Hewan coba dikelompokkan secara

random

K KI KI K K
I I II VI V

Aklimatisasi 7 hari

Timbang tikus sebelum sleep deprivation

PSD 4 TSD 4
Tidak diberi PSD TSD hari +
hari +
induksi stres 5 hari 5 hari sleep
sleep
recovery recovery
5 hari 5 hari

Lampiran 3. Tata Urutan Kerja

Timbang tikus setelah sleep deprivation dan sleep recovery
Tata Urutan Kerja

Pengambilan sampel darah retro orbital

Bius dan terminasi hewan coba

Pemeriksaan Superoxide dismutase dengan spektrofotometri

Dokumentasi

Pengolahan dan analisis

data
Lampiran 4. Pemeriksaan kadar SOD dengan metode spektrofotometri

Prosedur pemeriksaan SOD

1. Tentukan jumlah microtitter yang akan digunakan kemudian letakkan pada penahannya.
2. Pengambilan sampel darah tikus dilakukan setelah perlakuan sleep deprivation yang
dilakukan selama 96 jam dan dua kelompok lain setelah pemberian perlakuan sleep
recovery selama 7 hari.
3. Pengambilan sampel darah tikus dilakukan dengan menggunakan pipet kapiler
hematokrit yang diambil melalui vena pada pembuluh darah di mata (periorbital).
4. Sampel darah dimasukkan kedalam tabung mikrosentrifuse yang didalamnya telah
terdapat antikoagulan berupa EDTA 10%.
5. Sampel darah yang telah terkumpul disentrifuse selama 10 menit dengan kecepatan
3000 rpm.
6. Setelah terpisah antara plasma dan korpuskula, keduanya dipisahkan pada tabung
mikrosentrifuse yang berbeda.
7. Plasma sebanyak 20 μl plasma ditambahkan reagen BPA (Bradford Protein Assay)
sebanyak 200 μl dan H2O 780 μl. Blanko yang digunakan adalah 800 μl H2O.
8. Pembacaan absorbansi dilakukan menggunakan Spektrofotometer dengan panjang
gelombang 595 nm.
9. Didapatkan konsentrasi enzim Superokxide Dismutase (SOD) serum sampel darah tikus.