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Chapter 8 APPENDICIES APPENDIX-I Composition of Pelleted Diet for Rats

INGREDIENTS

Weight

1.

Wheat Flour

2.85 Kg

2.

Wheat Bran

2.85 Kg

3.

Dried skimmed milk Powder

2.00 Kg

4.

Soyebean Oil

0.50 Liter

5.

Mollasen

0.15 Kg

6.

Fish meat

1.50 Kg

7.

Salt (common).

0.05 Kg

8.

Vitamins/ Minerals/amino acids (Premix)*.

0.10 Kg

________________________________________________________ Total weight.

10.00 Kg

This diet is prepared at National Institute of Health (N.I.H), Islamabad according to the standard approved by the Universities Federation for Animals Welfare.

* Composition of premix is given in appendix II.

165

APPENDIX II Composition of Vitamins/ Minerals/amino acids (Premix) Mixed in the Diet, for Rats.

INGREDIENTS

Weight

1.

Vitamin A

10,000 IU/Kg*

2.

Vitamin D

5,000 IU/Kg

3.

Vitamin E

50 mg/Kg

4.

Choline

800 mg/Kg

5.

Methionine

500 mg/Kg

6.

Sodium chloride

5 g/Kg

7.

Dibasic Calcium Phosphate

9.5 g/Kg

8.

Zinc Sulphate

24 mg/Kg

9.

Potassium Iodide

3 mg/Kg

________________________________________________________ Total weight = amount of the premix added in 10 kg of the Pelleted diet prepared.

*amount added/ Kg of the diet prepared.

166

APPENDIX III Composition of High Sucrose Diet for Rats

Ingredients

Casein

DL-Methionine

High Sucrose Diet g/kg 200

3

Sucrose

650

Cellulose

50

Corn oil

50

Salt mix

35

Vitamin mix

10

Choline bitartrate

2

Sucrose = 68% Protein = 20% Fat = 12%

167

Appendix IV Pipetting protocol for incubation of cultured Leydig cells from rats CS = Cell suspension BIO PO4 = Biophosphate buffer Tube #

CS = 200 µl

1

Sample

2

Sample

3

Sample

4

Sample

5

Sample

LH 10 ng

6

Sample

LH 100 ng

7

Sample

LH 1000 ng

8

Sample

LH

9

Sample

LH

10

Sample

LH

11

Sample

12

Sample

13

Sample

LH = 50 µl

Drug

Drug

Visfatin10-6 30 µl Visfatin10-8 30 µl Visfatin10-10 30 µl

Visfatin10-6 30 µl Visfatin10-8 30 µl Visfatin10-10 30 µl Resistin10-6 30 µl Resistin10-8 30 µl Resistin10-10 30 µl

Drug

Drug

Drug

Drug

Bio PO 4 70 µl 70 µl 70 µl 70 µl 50 µl 50 µl 50 µl 20 µl 20 µl 20 µl 70 µl 70 µl 70 µl

168

14

Sample

LH

Resistin10-6 30 µl

20 µl

15

Sample

LH

Resistin10-8 30 µl

20 µl

16

Sample

LH

Resistin10-10 30 µl

20 µl

17

Sample

18

Sample

19

Sample

20

Sample

LH

21

Sample

LH

Rosiglitazone10-8

22

Sample

LH

Rosiglitazone10-10 30 µl

23

Sample

db cAMP 0.05 mM 30 µl

24

Sample

db cAMP 0.5 mM 30 µl

25

Sample

20 µl

Rosiflitazone10-6 30 µl Rosiflitazone10-8 30 µl Rosiglitazone10-10 30 µl Rosiglitazone10-6 30 µl

db cAMP 5 mM 30 µl -6

26

Sample

LH

27

Sample

LH

28

Sample

LH

29

Sample

Resistin10 30 µl Resistin10-8 30 µl Resistin10-10 30 µl

db cAMP 5 mM 10 µl db cAMP 5 mM 10 µl db cAMP 5 mM 10 µl Pregnenolone 0.03 µM 30 µl

70 µl 70 µl 20 µl 20 µl 20 µl 70 µl 70 µl 70 µl 10 µl 10 µl 10 µl 40 µl

169

30

Sample

31

Sample

32

Sample

LH

33

Sample

LH

34

Sample

LH

35

Sample

LH

36

Sample

LH

37

Sample

LH

Visfatin10-6 30 µl Visfatin10-6 30 µl Visfatin10-6 30 µl

Pregnenolone 0.3 µM 30 µl Pregnenolone 3 µM 30 µl Pregnenolone 3 µM 10 µl Pregnenolone 3 µM 10 µl Pregnenolone 3 µM 10 µl PKC blocker 20 µM 10 µl PKA blocker 20 µM 10 µl Ras / Raf1 kinase blocker 20 µM, 10 µl PKC blocker 20 µM 10 µl

PKC blocker 20 µM 10 µl

Resistin10-6 30 µl Resistin10-8 30 µl Resistin10-10 30 µl

38

Sample

LH

Visfatin10-6 20 µl

39

Sample

LH

Visfatin10-6 10 µl

40

Sample

LH

41

Sample

LH

42

Sample

LH

Rosiglitazone10-8 30 µl Rosiglitazone10-8 30 µl Rosiglitazone10-8 30 µl

PKC blocker 20 µM 10 µl PKA blocker 20 µM 10 µl Ras / Raf1 kinase blocker 20 µM, 10 µl PKC blocker 20 µM 10 µl PKC blocker 20 µM

43

Sample

LH

Rosiglitazone10-8 20 µl

44

Sample

LH

Rosiglitazone10-8

40 µl 40 µl 10 µl 10 µl 10 µl 10 µl 10 µl 10 µl PKA blocker 20 µM, 10 µl PKA blocker 20 µM, 10 µl

10 µl Ras / Raf1 kinase blocker 20 µM, 10 µl

10 µl 10 µl 10 µl 10 µl

PKA blocker 20 µM, 10 µl PKA

10 µl Ras / Raf1

10

170

10 µl

45

Sample

LH

46

Sample

LH

47

Sample

LH

Visfatin10-6 20 µl Visfatin10-6 20 µl

All tubes were run in triplicate.

Resistin10-8 20 µl Resistin10-8 20 µl

Rosiglitazone10-8 20 µl Rosiglitazone10-8 20 µl

10 µl

blocker 20 µM, 10 µl

kinase blocker 20 µM, 10 µl

µl

10 µl 10 µl 10 µl