· 2016. 11. 20. · 2 ก ก 2 ˇˆ ˆ 1 ˝˛ ˚˜ ก 2554 ก # $%&’( & &) * $+...

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1 กก 2 1 ก 2554 1: ก (Part 1: Wildlife nutrition)

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Page 1:  · 2016. 11. 20. · 2 ก ก 2 ˇˆ ˆ 1 ˝˛ ˚˜ ก 2554 ก # $%&’( & &) * $+ ,ก - .ก ,./ ก 0 + 12$( $ ก 3ก %) $%245, 65 5) *7 ก +/ 8ก2/9:) * 7

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������� 1: �� ��ก������� (Part 1: Wildlife nutrition)

Page 2:  · 2016. 11. 20. · 2 ก ก 2 ˇˆ ˆ 1 ˝˛ ˚˜ ก 2554 ก # $%&’( & &) * $+ ,ก - .ก ,./ ก 0 + 12$( $ ก 3ก %) $%245, 65 5) *7 ก +/ 8ก2/9:) * 7

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Evaluation of related nutrition management and nutrients quantity to breeding success in captive Endangered Pheasant (Great Argus) at Khao Kheow Open Zoo, Chonburi Province.

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2.4 �'*�ก�� (Methods) Eกe����>�K >I�]���E���I?�KRBN��T�IQ> �������ก�: ��E���I?���_�G����>I�@���?GQ��ก�����ก� Proximate analysis W�� ���ก>�GQ��@�>I�����P���E���I?G� ��S -R����� (Protein) ��E���I?RG���P� I����� Nitrogen �� Kjeldahl Method -]A��� (Fat) ��E���I?RG���P� Solvent extraction Method -E�Y�>T� (Crude Fiber) ��E���I?RG���P� Acid-Base extraction Method -��EW��� (Calcium) ��E���I?RG���P� Titration Method -p>@p>��@ (Phosphorous) ��E���I?RG���P� Vanado-Molybdate colorimetric Method -���NYS� I�Y>�SD (Moisture) ��E���I?RG���P� Drying Method -��?R�]oEG�� (Carbohydrate) ��E���I?GQ����P� by difference -�KC�� � (Energy) ��E���I?GQ����P� Calculation

*������������� Review ��ก �ก�!�"#$!�%&'#("���� )*+,!�$' -./�$� !%&����-0 2543-2549

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��ก������ก�ก����CX��ก���ก�E�Y>กก��>I�: NK� E�� 08.45-09.30 �. �กN�� >I��K��N��GT@K� T�_G>I� (_G�� 1 N��G) N�� �G�����ก�SDI��ก>I����TIQ ����D������>�K >I���S I�G 15 N��G ��S ��S��������TIQ��>Q >� TIQ�����Tก�QE��� ��@�G@K��ก�E��S� ����D������ก�� (E�Y�> �ก�����>I�RG�������D����Dก�G) NK� E�� 17.00 �. �G�����ก�SDI��ก>I�EI�Y> �D����K�����ก�ก��>I� (�K��N��G) �K>ก� �K>��� NK� E����I�K �����ก�CX��ก���ก�E�Y>กก��>I����J���A> ก�TIQE�Y>ก (Choice treatment) ��E���I?���������K� RBN�ก����]GQ���: �Dก���E���I?���������K� RBN�ก����@���?]GQ���GQ����P� Calculate by ratio Eกe������กAQ>�J����]GQ���Dก���E���I?AQ>�J�GQ����P�ก�� @_��� �� ANOVA RG�E�����E��������ก�K ��I�K �JK������ก�K ��I�K ก��K�ก��G�> GQ����P� LSD �� Duncan�s ���DG��

3. _2ก��[\กU� �กก���ก����@��C��P?��I�K ก���Gก�� GQ�RBN�ก�>I����KRBN�� ก��ก�EC�E��S� A��C��P�?�ก

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3.1 CX��ก���ก�E�Y>กก���������ก�ก��>I�A> �กI�QE������K>ก� �K>��� 3.2 �����RBN��T����_�G����>I�@���? 3.3 ���������K� RBN�ก�����กI�Q]GQ���T��K��NK� ��� 3.4 CX��ก���ก�A��C��P�?

3.1 )Y�'ก��&ก��%2`0กก'�12$#�'&�Pก��ก'�0�+��60,�ก+�/�%a2��5��0ก�,��0��� • ก��%2`0กก'� �กก���ก�GQ����P�ก���TIQE�Y>กก�� (choice treatment)RG�ก�TIQ>I����E�f����_�G��I�Y>>I�

@DE�e��J����c@�T�@J��>I�@���?��ก �D���ก�K 15 N��G �Q���ก�N��G�������>I�����กI�QE�Y>กก��C��KT�NK� 30 ����ก >I�����กI�Q�����_��T�ก�E�Y>กก��กK>��ก���@�G�Y> > �K�����S I��GW�� �����_��T�ก�E�Y>กก��กK>��ก�กI�Q�K��ก� �ก���@�GE�f�>��G���ก �ก�K �ก>I�N��G>Y��V >�K �����@D��U� @_��� ( P <0.05 ) �> � ��Y> ก�Q�� ����ก> AQ�ก�Q> ���AQ� >I�@���A >I��กE�eGG @K��>I����EB�>Y��V ENK� AQ�p= AQ�E��Y>ก ��>I�]กK ��_Jกก��E�f��DG��@�G�Q�

• #�'&�Pก��ก'�0�+�� �กc�ก���ก������ก�ก��>I�A> �กI�QT��K��NK� ���C��K�กI�Q�K��ก� �������ก�ก��

>I�E�����RG����Tก�QE��� ก�� (�ก�K ก��>�K ]�K�����@D��U� @_��� P> 0.05, n=30 (ANOVA F=0.753, P=0.531)) W�� �������ก�ก��>I�]GQ��S �K����� 97.45 - 263.35 ก����K>����K>��� RG��กI�Qก� 17 ���������ก�ก���ก���@�G �> � ��Y>�กI�Qก� 11, ก� 18 ��ก� 15 E�f��DG��@�G�Q����DG�� (Mean±SD = 165.29±46.96 g) ��S ��S����E>��G�����ก�ก��>I�E�����A> �กI�Q@��_@G ]GQG� �� ��� 1

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����,��� 1 @G �����ก�ก��>I�T��กI�Q (Great Argus) �K>ก� �K>��� (Mean for groups in animal as feed per 1 day.)

N��G�ก (Common name)

ก� I��E�A (Number)

Daily feed intake per cage per 1 day �D��� ( N) mean± SD ( g )

�กI�Q GA( 18 ) 9 153.27 ± 52.41 a GA( 17 ) 9 179.87 ± 45.95 a GA( 15 ) 6 150.93 ± 35.14 a GA( 11 ) 6 175.79 ± 52.53 a

Total / mean 30 165.29±46.96

I��EI�� a, b �� c @G ���EI�Y>�I�Y>����ก�K ��I�K ก��K�>�K �����@D��U� @_��� E�Y�>E�����E��������ก�K �KE�������I�K �JKA> I�K��ก��G�> RG���P� LSD �� Duncan�s �����G�����ENY�>���� 95 %

��S ��SE�Y�>C����_� �����ก�ก����N��G>I��Q� C��K���EB�>I����_Jกก��RG�E������ก�กI�Q

�ก���@�G�Y>>I����EB�N�K��SD ENK�c�]�Q�K V 42.74% ��ก��K�P�UCYN 28.70 % �> � ��Y>AQ�ก�Q> I� c@�]AK�Q� 18.15 % ���GQ��>I�@DE�e��J� 9.94 % ��>I���N���� 0.47 % W�� �>กEI�Y>�ก��S I��G��I�>�����������ก�TIQ�Q>��Q� >I�]กK]AK@DE�e��J� ��AQ�E��Y>ก�กEA���������ก�ก��RG�E������ก�กI�Q�Q>����@�GEC�� �Q>��� 1.47 �� 1.33 ���DG��E�K��S� (E�Y�>��G�ก�����>I���S I�G) _� �Q���������ก�TIQT�>���@K�����@J กe�� ��S ��S��AQ>@� Eก��K�����ก�ก��>I�I�Y>���_�G��� N��G>�_Jก�Dก�G�ก�����ก�TIQ������D����Q>� ENK�>I��DC�ก > �K� ,��S I��G ��I�>�

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Materials ,Food and Products

Nutrient Composition per 100 g

Proximate Composition Minerals

Energy Moisture Fat Protein Ash Fiber Carbohydrate Calcium Phosphorous

Kcal grams milligrams

ก�Q�� Banana 124.61 68.44 0.21 0.87 0.67 2.30 29.81 22.10 34.20

����ก> Papaya 30.40 92.22 0.04 0.44 0.23 0.50 7.07 15.60 8.50

AQ�E��Y>ก Paddy grain 352.41 7.40 2.05 7.21 7.06 9.87 76.28 162.30 315.20

AQ�RCG Corn ( yellow ), raw 86.57 77.96 0.17 3.01 0.61 0.84 18.25 16.60 19.00

>I�]กK]AK Hen food 358.82 4.40 5.18 18.38 12.37 5.50 59.67 3638.70 604.90

AQ�ก�Q> +]AK]กK�Q� Eggs unpolished rice, steamed 113.49 72.84 1.37 3.86 0.50 0.60 21.43 30.00 110.00

I�>� Meal worm 165.40 66.20 7.00 17.30 1.20 5.70 8.30 20.00 200.00

>I��กE�eGG bird food 374.70 7.96 6.94 25.39 7.04 0.67 52.67 1167.80 1009.70

>I�@���A puppy food 399.61 3.70 8.69 26.40 7.26 3.50 53.95 1480.00 1290.00

AQ�ก�Q> Rice ( unpolished ) 370.24 9.03 2.20 7.33 1.16 2.40 80.28 23.20 272.50

AQ�p= Millet 370.81 8.31 3.09 9.70 2.85 8.90 76.05 183.60 343.00

���AQ� Rice ( broken ) 366.12 9.32 1.24 7.70 0.70 0.50 81.04 465.00 134.30

> �K� Grape 38.99 89.46 0.15 0.55 0.98 1.10 8.86 20.00 20.00

��S I��G Cricket 174.30 67.80 9.98 17.70 1.10 5.40 3.42 9.00 151.00

ก��G> ��I��ก Shell of squid - 34.78 0.00

3.3 #�'&�P(7P(����,�� ��ก������ก+�/�T./��38�1��2$ ��,��� �กก���ก����������K� RBN�ก�����กI�Q �D������ 8 ��� (4 ก� ) ]GQ���T��K��NK� ��� RG�@G T�

�J�A> �KC�� ���S I�G ���Q>���E������K>>I�@G 100 ก��� (Average per 100 g edible as feed) C��K����K� RBN�ก��K V ���]GQ�����S����ก>�GQ��

• (��)2�,,�� (Gross energy: Kcal) �กก���E���I?�����C�� �T�>I�����กI�Q�K��ก� ]GQ���T��K�������S�C��K RG����]GQ����K

C�� ���S I�GE����� (Mean ±SD) E�Kก�� 232.72±80.54 ก�R���>�� (Kcal) �K>ก� �K>��� RG�T��D����กI�Q��S 4 ก� ��S�C��K�กI�Qก� 15 (ก� ���A��C��P�?]GQ) ]GQC�� ��ก���@�G 271.85±73.88 Kcal �> � ��Y>�กI�Q ก� 11 (267.92±91.62 Kcal), ก� 18 (210.89±78.42 Kcal) ��ก� 17 (205.00±72.31 Kcal) ���DG�� W�� E�Y�>C����E�����E����GQ����P�ก�� @_����Q�C��K�KC�� �RG����������ก�K ก��>�K ]�K�����@D��U� @_��� (P> 0.05, n=30 (F=1.505, P=0.237))

• (��#�'&�P(��& ̀4� (Moisture: %#0��%hi���)

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�กก���ก�_� ��������NYS�T�>I�����กI�Q]GQ��� RG�C����T��J���A> ������ก@�G@K��>I�I��ก��กV 100 ก��� (Average per 100 g I�Y>�Q>���A> �SDI��ก@G) ����กI�QE�Y>กก����S� C��K����������NYS�RG�E�����E�Kก�� (Mean ±SD) E�Kก�� 64.97±7.80 E�>�?EWe��? W�� E�Y�>C����E�����E������������NYS�����กI�QT��K��ก� ]GQ�����S�C��K RG�����กI�Q��S 4 ก� ]GQ������NYS�T����������ก�K ก��>�K �����@D��U� @_��� (P> 0.05, n=30 ( ANOVA F=11.964 , P=0.000 )) W�� �กI�Qก� 17 E�Y>กก��>I��������������NYS�@J ���@�G _� 71.95 % @K���กI�Qก� 15 E�Y>กก��>I��������������NYS��Q>����@�G EC�� 55.56 %

• (��#�'&�PT6&�� (Fat: %#0��%hi���) T�@K��A> �����]A�������กI�QT��K��ก� ]GQ�����S�C��K �กI�QRG����]GQ���]A���E�����( Mean

±SD )E�Kก�� 1.35±0.30 E�>�?EWe��? (�ก>I� 100 ก���) �K>ก� �K>��� W�� �กI�Qก� 15 (���A��C��P�?]GQ)]GQ��������]A����ก>I����E�Y>กก��EAQ]��กก�K�กI�Qก� >Y��V ���EI�Y> (ก� 18,17 �� 11) >�K �����@D��U� @_��� (P< 0.05)RG�]GQ���]A���E�Kก�� 1.60 E�>�?EWe��? ��S ��S�กI�Qก� 17 ���]�K��CX��ก���ก�c@�C��P�?� ]AK ]GQ���]A����Q>����@�G ��GE�f� 1.16 E�>�?EWe��? W�� RG�����Q�C��K�����]A�������กI�Q�K��ก� ]GQ���������ก�K ก��>�K �����@D��U� @_��� (P< 0.05, n=30 ( ANOVA F=3.219 , P=0.039 ))

• (��#�'&�P�#���� ( Protein: %#0��%hi���) �กก���E���I?�����R����� C��K�กI�Q]GQ���R�����E����� (Mean±SD) 4.85±1.07 E�>�?EWe��? �K>

ก� �K>��� W�� E�Y�>C����E�����E���������R���������กI�Q�K��ก� ]GQ���C��KRG����������ก�K ก��>�K �����@D��U (P<0.05, n=30 (ANOVA F=4.81, P=0.009)) ��S ��S�กI�Q ก� 15 ]GQ��������R������ก���@�G (Mean±SD = 5.92 ± 1.13 %) ENK�EG���ก��ก���KC�� ���]A��� W�� �กก�Kก� 18 �� 17 >�K �����@D��U (P<0.05) �KE�Y�>E����ก���กI�Qก� 11 C��K�ก�K >�K ]�K�����@D��U� @_��� (P>0.05)

• (��#�'&�P%W/� (Ash: %#0��%hi���) �กก���E���I?�����E_Q��>���@K��>I�����กI�Q]GQ��� C��K�������E����� E�Kก�� 1.41±0.36

E�>�?EWe��? �K>ก� �K>��� W�� RG����������ก�K ��I�K ก� >�K ]�K�����@D��U� @_��� (P>0.05, n=30 (ANOVA F=4.81, P=0.009)) �กE�Q��กI�Qก� 15 W�� ]GQ��������E_Q�ก���@�G (1.65 %) ��E�Y�>E�����E����ก���กI�Qก� 17 W�� ]GQ���E_Q�Q>����@�G (1.25 %) ��������ก�K >�K �����@D��U� @_��� (P<0.05)

• (��#�'&�P%5̀�085 (Crude fiber) T�@K��A> �����E�Y�>T�T�>I�����กI�Q]GQ��� C��K�กI�Q]GQ���E�Y�>T�E����� 1.51±0.40 E�>�?EWe��? �K>

ก� �K>��� �����E�Y�>T�T��กI�Q�K��ก� RG����������ก�K ก��>�K �����@D��U ( P< 0.05 , n=30 ( ANOVA F=5.026 , P=0.007 )) RG��กI�Qก� 15 ]GQ���E�Y�>T��ก���@�G1.87 % �> � ��Y>ก� 18 (1.61%), ก� 11 ( 1.43 % )��ก� 17 �Q>����@�G 1.22 % ���DG��

• (��#�'&�P(����3Tj%.�� (Carbohydrate) �กก���E���I?I�������?R�]oEG������กI�Q]GQ����กก�ก�� GQ����P�ก��� by difference C��K

RG�����กI�Q]GQ�����?R�]oEG�� E����� 35.26±3.44 E�>�?EWe��? �K>ก� �K>��� W�� E�Y�>C����E�����E����T��กI�Q�K��ก� C��K������ก�K ก��>�K �����@D��U� @_��� (P< 0.05, n=30 (ANOVA F=14.39, P=0.000)) ��S ��S �กI�Qก� 15 ]GQ�����?R�]oEG���กก�ก��>I��ก���@�G _� 35.26±3.44 E�>�?EWe��? �K>��� W�� �กก�K�กI�Q��กก� ���EI�Y>>�K �����@D��U (P < 0.05) �>ก�ก��SC��K�กI�Qก� 17 ]GQ�����?R�]oEG���Q>����@�G �ก�K �ก��กก� ���EI�Y>>�K �����@D��U (P < 0.05)

• (��#�'&�P1(2%h�5& (Calcium)

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�กก�������G������KP����EW��� (Ca) ����กI�Q]GQ��� C��KRG�����กI�Q]GQ�����EW���E����� (Mean±SD) E�Kก�� 0.20±0.00 E�>�?EWe��? �K>ก� �K>��� W�� E�Y�>E�����E�����������EW������]GQ���T��K��ก� C��K]�K������ก�K ก��>�K �����@D��U� @_��� (P > 0.05, n = 30 (ANOVA F=0.137, P=0.937))

• (��#�'&�Pk0�k0��� (Phosphorous) �กก�������E���I?������KP��p>@p>��@ (P) ����กI�Q]GQ��� C��KRG�����กI�Q]GQ���p>@p>��@

E����� (Mean±SD) E�Kก�� 0.17±0.00 E�>�?EWe��? �K>ก� �K>��� W�� E�Y�>C����E�����E���������p>@p>��@���]GQ���T��K��ก� C��K������ก�K ก��>�K �����@D��U� @_���( P < 0.05, n = 30 (ANOVA F=6.317 , P=0.002 )) RG��กI�Qก� 15 ]GQ���p>@p>��@@J ���@�G 0.21 % �กก�Kก� >Y��V >�K �����@D��U� @_��� ( P< 0.05 )�> � ��Y>�กI�Qก� 11 �� 18 W�� ���KTก�QE��� ก�� @K���กI�Qก� 17 ]GQ���p>@p>��@�Q>����@�GEC�� 0.13 % �Q>�ก�Kก� >Y��V ��กก� >�K �����@D��U� @_��� (P < 0.05) ����E>��Gc�ก���E���I?����K� RBN�ก�����กI�Q]GQ���@G ]GQG� �� ��� 3 �� 4 ����,��� 3 @G �����C�� �����กI�Q�K��ก� ]GQ���E������K>��� (Means for Nutrition description of energy value, calculate from animals as feed average per 1 day)

Proximate composition

ก� I��E�A (Number)

Nutrient Composition per 1 day �D��� ( N) mean± SD (Kcal )

Energy GA Cage18 9 210.89 ± 78.42 a GA Cage17 9 205.00 ± 72.31 a GA Cage15 6 271.85 ± 73.88 a GA Cage11 6 267.92 ± 91.61 a

Total / mean 30 232.72 ± 80.54

I��EI�� a, b, c �� d @G ���EI�Y>�I�Y>����ก�K ��I�K ก��K�>�K �����@D��U� @_��� E�Y�>E�����E��������ก�K �KE�������I�K �JKA> I�K��ก��G�> RG���P� LSD �� Duncan�s �����G�����ENY�>���� 95 %

����,��� 4 @G ���������K� RBN�ก�����กI�Q�K��ก� ]GQ���E������K>��� (Means for Nutrition description of proximate composition and Minerals, calculate from animals as feed average per 1 day)

Proximate composition And minerals

ก� I��E�A (Number)

Nutrient Composition per 100 g edible feed �D��� ( N) Mean± SD (%)

Moisture GA Cage18 9 66.04 ± 4.67 b GA Cage17 9 71.95 ± 5.46 c GA Cage15 6 55.56 ± 5.01 a GA Cage11 6 62.29 ± 6.37 b

Total / mean 30 64.97 ± 7.80

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Proximate composition And minerals

ก� I��E�A (Number)

Nutrient Composition per 100 g edible feed �D��� ( N) Mean± SD (%)

Fat GA Cage18 9 1.32±0.21 ab

GA Cage17 9 1.16±0.25 a GA Cage15 6 1.60±0.37 b GA Cage11 6 1.41±0.30 ab

Total / mean 30 1.35 ± 0.30

Crude Protein GA Cage18 9 4.66 ±0.84 a GA Cage17 9 4.16 ±0.78 a

GA Cage15 6 5.92 ± 1.13 b GA Cage11 6 5.10±0.94 ab

Total / mean 30 4.85 ±1.07

Ash GA Cage18 9 1.43±0.19 a

GA Cage17 9 1.25±0.54 a GA Cage15 6 1.65 ±0.13 a GA Cage11 6 1.39±0.29 a

Total / mean 30 1.41 ± 0.36

Crude fiber GA Cage18 9 1.61 ± 0.23 bc GA Cage17 9 1.22 ± 0.37 a GA Cage15 6 1.87 ± 0.29 c GA Cage11 6 1.43 ± 0.49 ab

Total / mean 30 1.51 ± 0.40

Carbohydrate GA Cage18 9 26.55 ± 3.77 b GA Cage17 9 21.48 ± 4.23 a GA Cage15 6 35.26 ± 3.44 c GA Cage11 6 29.80 ± 4.94 b Total / mean 30 27.42 ± 6.34

Calcium GA Cage18 9 0.20 ± 0.00 a GA Cage17 9 0.20 ± 0.14 a GA Cage15 6 0.21 ± 0.00 a GA Cage11 6 0.22 ± 0.00 a Total / mean 30 0.20 ± 0.00

Phosphorous GA Cage18 9 0.17 ± 0.00 b GA Cage17 9 0.13 ± 0.00 a GA Cage15 6 0.21 ± 0.00 c GA Cage11 6 0.17 ± 0.00 bc Total / mean 30 0.17 ± 0.00

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N��G>I� (Food diversity) ����กI�Q]GQ���T��K��NK� XGJก� ��I�K NK� A> ก���ก��� 2553 �� 2554 C��K������ก�K ก�� RG��กI�QT�NK� XGJก�A��C��P�? �� 2554 ]GQ���>I�������KG�N�����I�กI���กก�KT�NK� XGJก�A��C��P�? �� 2553 E�eก�Q>� RG����KG�N�����I�กI�� ��I�K �� 2554 �� 2553 E�Kก�� 2.69 �� 2.59 ���DG�� ����E>��G@G G� �� ��� 2 �� 3 ����,��� 2 @G AQ>�J�� GQ�N��G>I������I�กN��G���TIQ�กI�QB�T�@��@���?v

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G�N�����I�กI�� (Food diversity) 2.59 2.69 ����K� RBN�ก�

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Robinson, J.J. 1996. Nutrition and reproduction. Anim. Reprod. Sci. 42:25-34. R. Plame, S. Rettenbacher, C. Touma, S. M.El-Bahr, and E. Mostl. 2005. Stress Hormones in mammals and birds

Comparative Aspects Regarding Metabolism , Excretion, and Noninvasive Measurement in Fecal Samples. Ann. N.Y. Acad. Sci. 1040: 162 171.

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�GE�f�@���?>�กก��K�I��� �����G�K�����@D��U�K>> �?ก� E�Y�> GQ��E�f�@���?E��S� �JกGQ�������>���>�JK���ก�����A�GTIUK@�G�> � ��กNQ �r���������GEI�Y>>�JKT�R�กEC�� 5 N��G ��กN��G��G>�JK� �? ]�R�EW>R���G� (Family Rhinocerotidae) ���������@���?�DC�ก�GEAQ]�QGQ��ก�� W�� E�f� 1T� 3 � �?A> ก��K�@���?ก����� (Odd-toed ungulates) �����T�R�ก RG��GA������>�JKT����GJ�A> @��@���?E�FGEAEA�����S� ��GE�f��G���TIUK���@�GA> @���?ก��K���S �GA����GN��G>Y��V E�f�@���?������D���EI�Y>>�JKT�P���N��]�K�ก��ก>�ก��S >�JKT�@B��Tก�Q@JUC��P�?E�Y�> �ก_Jก�K>�K I��ก �GT�P���N���� >�JKT�B��>�����_� �QT��r������c��กก�GDE���ก��K V ECY�>�t> ก��ก���ก�>��K@���?��@K c�TIQ�D������Nก��GA> BJ��B��K V >�JKT���G������ ��� �Kก�GDE������ก�>����ก�?�>ก_���>��� (ex situ) ECY�>�t> ก��ก�@JUC��P�?กe�� � �����@D��U���Q> GDE���ก��K>]� >�ก��S �� E�f����R�N�?�K>ก�TIQก���ก�กK@P���N���Jก<r _� ���@D��UA> N��GC��P�?��ก��t> ก��_���>��� ก�>����ก�?ECY�>TIQEก�G������ �Y�A> ���Nก�@���?]GQGQ����E> ��S�������DE�f��Q> EC�����G��A> >���ก�A��C��P�? C�Q>�ก����S�Q> �G>���ก���� �Kก�EC�E��S� ECY�>TIQA��C��P�?]GQT�ก� E��S� ��S�E�f�E�Y�> ����DTIQ���@����@DE�e�]GQ�กE�Y�> �กAGAQ>�J����@D��UI�����ก� ��S ��SECY�>�>�@�> �K>C��P�ก��G� ก�K�� @��@���?v �� ]GQ��E����GDE���ก���ก��rUI���Eก����AQ> ก��A��C��P�?���ก>�ก��ก���ก������กK>�I�Q��SRG� >����ก ����� (2552) ���]GQ�Dก�C�@J��?@�����u�_� ���E�f�]�]GQT�ก���S �Q> GQ��ก�������G�����o>�?R��R��E�@E�>R�� (Progesterone) T�>�����A> �GA�EC�E������>�JKT�@BCA> ก�EC�E��S� A> @��@���?E�FGEAEA��� �D��� 2 ��� (NY�>@���� ��A���) GQ����P�ก�������E���I?�� ELISA ��I�K NK� EGY>� P����� 2551 _� EGY>������ 2552 W�� C��K�����o>�?R��R��E�@E�>R�� ���������G]GQ>�JKT�NK� 0.04 _� 1.05 µg/g of dry feces W�� @G _� ��R�Q����]�K��ก�E�������� E�f�� �>� (non cyclic) ������������EAQ�AQ���D >�ก��S @G _� B�����]�K]GQ��ก���S �Q> I�Y>E�f�@�G T�NK� ����E��G� ก�K�. �r������@��@���?E�FGEAEA�����ก�EC�E��S� �GA�EC�E�����EC�cJQ�����S @�S� 4 ��� ��S ��ST�NK� �� 2552@��@���?E�FGEAEA���]GQ����GA�EC�cJQEAQ��G��EC�cJQ������ ก���EC�cJQEAQ����ก��K�ก��EC�E���EG����S��DTIQEก�G@�����u�_� ���E�f�]�]GQT����@��_�����EI������DTIQ�GA�EC�E�������G��A> o>�?R���K V @J A�S��กEG����>��DTIQEก�GB��A> ก�E�f�@�G]GQT��GA�EC�E��� �>ก�ก��S�� C��K��CX��ก���ก�c@�C��P�?ก��T�ก��K�A> �GA����>�JKT�@BCA> ก�EC�E��S� T�NK� �Q��� 2553 �� ��@�����u�EC���E���_� ก���S �Q> A> �GA�EC�E�����S @> ��� ������ <=�>����ก�?�������W�� TIQ���@D��UA> ก���G� ก�K��� ]GQGDE���ก�Eกe����>�K �J�A> �GA�ECY�>�D�������E���I?�����o>�?R��>�ก���S >��E�f�ก��G@>�_� @�����u���S I��G� ก�K� ECY�>���R�N�?T�ก�������� C�{�ก���Gก�ก� E��S� TIQ�����@��P�BC�กA�S��K>]�. 1.1 -7.&7�,+&�58�ก��[\กU�

1. ECY�>���E���@BC� @�������A> ����@Y�C��P�?��E�����E����ก�E���UC��P�?A> ���Nก��GA�EC�E���T�@��@���?E�FGEAEA���

2. ECY�>��ก�ก�E�������� A> ��G��o>�?R��R��E�@E�>R�� RG���P�ก�@ก�G�ก>����� 3. ECY�>TIQ���AQ>�J����E�f����R�N�?�K>ก�EC�A��C��P�?�GT�@��@���?E�FGEAEA���

1.2 (��&�V�(�:#�$�5 �����(�.���-$T./��3-�กก���'-�5 ก���ก����S ��SE�f�AQ>�J�CYS�u����@��_�D]������ก�?TNQ���R�N�?]GQI�กI�� >��ECY�>C�@J��?���

���@��C��P?��I�K o>�?R��ก��CX��ก���C>V ก��ก��ก��D�TNQกQ]A�>��rUIEก����ก������@Y�C��P�? (Reproduction) , @��@G�BC@���? (Animals welfare) , ก�>����ก�?���C�ก�� N��BC (Conservation) ��ก���ก�GQ����_�� N�����������

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�D�TNQ������GT�EN� �����_� ���@DE�e�T�ก�������� ก���Gก�ก� E��S� ECY�>TIQ@���?��CX��ก���ก�A��C��P�?��P���N��]GQ

�>ก�ก��S�� E�f�ก�C�{���ก�BC��ก�����TIQ�����ND�UT�ก�TNQE������K V T�ก�@ก�G����E���I?o>�?R��� N��G�ก�J�@���? >�ก��S �� @��_TNQI��กก�EG���ก����Sก��@���?�=�K V ���@D��U��Tก�Q@JUC��P�?T�>����K>]�.

1.3 ก�031��('. �YUZ����8 /8�ก���'-�5 ก���ก���]�K�Dก������ ���I�Y>��ก�����@���? (Noninvasive methods) RG�ก�������G�����A> @E����>�G?o>�?R��T����>�K �J�@���?��S�E�f���P�ก�I��� ��������EI��@�@DI���@���?���>�JKT�@BCA> ก�EC�E��S� (Rupert et al., 2005) ��S ��SECY�>I��กE���� c�ก������>ก��GE�e����>���Eก�GA�S�ก��@���?T�B�I�� RG�>���I��กก�����K �o>�?R��� _Jก@�Q �ก�����K>�]�Q�K>�K V ��_Jก@K EAQ@JKก��@E�Y>GECY�>cK�]��� >�����E�tI�� �ก��S���_JกI���E����cK�EAQ]�B�T����W�� ��_Jก metabolized E�������� @BCE�f�>��C��P?�K V �ก��S���_Jก@K �K>EAQ�D]@QT��J�A> �SDG�W�� ��EAQ@JKก�����ก�Ecc�U>I�A> �K ก� (metabolites) ��_JกA��_K�>>กRG����ก��>����� W�� ����ก�E���ก���V @E����>�G?��S I�G���>�JKT�>������K �Fecal steroids� (Erich et al., 2005)

ก�����@E����>�G?T�>������� @��_GDE���ก�ECY�>E�f�ก����E���@B��@BCA> �����K>�]�Q�K>]GQ W�� ����G��S_Jก��E����A�S��T�NK� ������@�?��ก�N 1970 �����ก�T��ก ��NK� �Q����@�?��ก�N 1980 ��ก�T�@���?E��S� �JกGQ���� (Rupert, 2005; Wolfgang et al., 2005) �DG�����@D��UA�S�@J A> ก�������D���EAQT�@_�BCA> ก�@Y�C��P�?T�@���?���>�JKT�@BCA> ก�EC�E��S� ���r�����K V �����>��P�C��K>���@��J��?C��P�?��S��Y>ก��D���EAQT�_� o>�?R�� o>�?R���Y>�r����T�ก�A��E��Y�>�@JKก�����ก�@Y�C��P�?���J���A> ก�I��� o>�?R���K V @��_TNQE�f�G�N��N�S��G@B��@BCA> ก�@Y�C��P�?]GQ (Janine et al., 2002) AQ>@� Eก����_Y>E�f����กQ�I�QG� ก�K�]GQ_JกGDE���ก�A�S�GQ��ก���ก�_� �J���ก��D �A> �� ]AK(ovarian function )W�� ก���ก����S �กV �Y>ก���E���I?����� progestogen metabolites ���_JกA��_K�>>ก�T��GGDEC�E���A> >p��ก (Schwarzenberger et al., 1998) �J���I�Y>��P�A> ก���E���I? (assay methodologies) ���_Jก�D�TNQT�ก���ก���S�����S enzyme immunoassays �� radioimmunoassay ���TNQI��กA> ก��D���ก������ cross react A> broad-spectrum antisera ก��>��C��P�?�K V A> pregnanes (Janine et al., 2002) RG�A�S��กA> ก���ก����]GQGDE���ก���S�Eก����AQ> ก��ก�E�����E���������o>�?R��EC����_JกA��_K�>>ก� (gonadal steroid metabolites) A> @���?�K��������>�JKT�ก��K����Nก�A> @��@���?E�FGEAEA���

2. �'*�ก��[\กU� 2.1 07#ก�P� (Materials Essential Component of the EIA )

• Solid Phase: polystyrene microtiter plate (Nunc Immuno plate) • Monoclonal Antibody: Progesterone CL-425 • Coating buffer: Carbonate/bicarbonate buffer pH 9.6 • Wash solution: NaCl and Tween 20 solution • Enzyme conjugate (tracer): Progesterone Horseradish peroxidase (HRP) • Assay buffer: Phosphate or Tris buffers pH 7.0 • Standards or unlabeled antigen: Standards Progesterone (Sigma Diagnostics) • Substate: Chromagen (ABTS ) and catalyst (Hydrogen peroxide) Substrate

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• Reading: Spectrophotometer or Plate reader 2.2 ���05��,����� (Animals sample)

• �GA�EC�E��� �D��� 2 ���

2.3 �'*�ก�� (Methods) �Dก���ก�T��GA�EC�E��� �D��� 2 ������>�JKT�������c�GN>�A> @��@���?E�FGEAEA��� RG���ก�Eกe����>�K >����� >�K �K>E�Y�> ��I�K NK� EGY>��ก��� 2552 E���� 2553 T@KT�ก�K> Eกe����>�K ���D]�Eกe���ก� ECY�>�>ก�������E���I?o>�?R��EC�E�����ก���S �Q> �Y> o>�?R��R��E�@E�>R���ก� IQ> �������ก� ����]�K�Dก���ก��@���?]�K�Kก���TGV ��>G����E������Dก������ RG���A�S��>���K�V G� ��S�Y> Eกe����>�K >������GA� (Collection feces): @�K�Eกe����>�K >�����A> �GA��K����� (20-50 ก���) @��GI?�� 1-5 ���>�K (�กE�Q�NK� EGY>�ก��BC��P?-E���� 2553 ���Eกe����>�K ]GQ�Q>�) E�Y>กEกe��K���>�K >������������ก���E�f�E�YS>EG���ก��ECY�>TIQ]GQ@E����>�G?o>�?R�������ก�ก�������ก��>�K @��DE@�>T����>�K RG�E�Y>กEกe�E�C�>������������ก���@GTI�K ]�KIQ �>ก�ก��S���>�K >��������@���?A��_K�>>ก�T�� ���S IกC��K>I��� _Jก�K>�]�KG�RG��� � �J�A> N�S�>I�I�Y>@�A> >I����ก��EAQ]�>�JKกe���Q> ��G�ก>>ก]�K�Dก�Eกe� RG�I��ก�Q��Q> E�Y>กEกe�E�C����>�K �������ก���E�f�E�YS>EG���ก��A> ���>�K >����� ��]�K��E����@G�>Y����>�JKT����>�K ก�Eกe���ก����>�K I�Y>�DTIQIQ (Preservation with Freezing or Drying immediately): ก�������� ]�K@��_�D���>�K TIQIQ ]GQ��� @��_�Dก�Eกe���ก����>�K ]�QT�ก�K> ���@ �����<��GECY�>�t> ก��]�KTIQ���>�K @��c�@ก�����NYS�I�Y>@ �ก��S��D]�Eกe�]�QT��JQNKAe >��IBJ�� -20 > �EW�EW��@ ECY�>Eกe���ก�@BCA> >������ก����E��� ��E�f�ก��t> ก��]�KTIQ@E����>�G?��ก�E�������� @BC]� �D���>�K ���NKAe Eกe���ก�@BC��DTIQIQ GQ��ก�]�K���NYS�>>ก�ก���>�K GQ��ก��DEAQ�JQ>� (Hot air oven) E�f�����E��]�K�Q>�ก�K 96 N���R� ���>��IBJ�� 60 > �EW�EW��@ ���SDI��ก]�KE�������� >�ก �ก��S�Eกe���ก����>�K GQ��ก�T@KI��IK>���@ T@K@�GJG���NYS�� ]�ECY�>�t> ก�����NYS� �FG<TIQ@����D]�Eกe�]�QB�T��JQ������>��IBJ�� ���E�e����YG ECY�>�t> ก��ก�E�������� A> @E����>�G? (Erich et al., 2005;Tony et al., 2005) �Q��>�D]�@ก�Go>�?R���K>]� ก�@ก�G@E����>�G?o>�?R���ก>����� (Fecal Extraction):

�Dก�@ก�GGQ����P�ก��Q� (Dry and Wet Weight Fecal Extraction Boiling Method) TNQ���>�K >������GA� 0.1 g @ก�GGQ�� Ethyl alcohol 90 % �Q�Eกe�@��������]GQ]�QT� dilution buffer NKAe ���>��IBJ�� -20 > �EW�EW��@ ECY�>�>ก���E���I?�K>]� ก���E���I?�����o>�?R�� (Assay Protocols): �Dก���E���I?GQ����P� Enzyme immunoassay �� Competitive ELISA (Enzyme Linked Immuno Sorbent Assay) ��ก�����ก�A> Janine et al.(2004)

3. _2ก��[\กU� �กก����E���@B��ก��D �A> �K>�o>�?R��EC�T�ก��K��GA�( EC�E��� )A> @��@���?E�FGEAEA���RG���P�ก���ก���]�K����� ���I�Y>��ก�����@���? GQ��ก�������E���I?��G�����EAQ�AQ�A> o>�?R��R��E�@E�>R��

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(Progesterone metabolites) T����>�K >����� (feces) A> �GA����>�JKT�@BCA> ก�EC�E��S� ����G@G ��S �K EGY>��ก���_� ����� 2552 ��EGY>�ก��BC��P?_� E���� 2553 ]GQc��D�ก���K������� �K>]���S

1. �GA� EC�E��������� 1 NY�> �A���� >�� 17 �� Global Studbook#: 1303 2. �GA� EC�E��������� 2 NY�> �@����� >�� 18 �� Global Studbook#: 1304 ก���ก�T�NK� EGY>��ก���_� ����� 2552 • �GA�EC�E���NY�> A���

�กก�������G��G��A> o>�?R��R��E�@E�>R�� (Progesterone metabolites) T����>�K >����� ��S �KNK� EGY>��ก��� 2552 _� ����� 2552 C��K �GA�NY�> �A���� ����G�����EAQ�AQ�A> o>�?R��R��E�@E�>R��E����� (Mean± SD) E�Kก�� 0.16 ± 0.1 µg/g of dry feces

• �GA�EC�E���NY�> @���� �กก�������G��G��A> o>�?R��R��E�@E�>R�� ( Progesterone metabolites) T����>�K >����� ��S �K

NK� EGY>��ก��� 2552 _� ����� 2552 C��K �GA�NY�> �@����� ����G�����EAQ�AQ�A> o>�?R��R��E�@E�>R��E����� (Mean ± SD) E�Kก�� 0.18 ± 0.1 µg/g of dry feces

�กก���ก�T��GA�EC�E�����S @> ��� C��K��G�����EAQ�AQ�A> o>�?R��R��E�@E�>R�����������G]GQ���KTก�QE��� ก�� ��S ��S]�KC�����ก�K >�K �����@D��U� @_��� �K��G�����EAQ�AQ�A> o>�?R��R��E�@E�>R��RG�E����� (Mean ± SD) A> �GA����NY�>�A���� @J ก�K �@����� E�eก�Q>� [(P>0.05 , n=201 (ANOVA F=1.216, P = 0.272)] RG����KE����� (Mean ± SD) ���C�E�Kก�� 0.17±0.1 µg/g of dry feces (C�@�� 0.03-0.62) W�� ����������EAQ�AQ���D]�K@G _� ก�E�������� E�f�� �>� (BC��� 1-2) ��]�K@G _� B��A> ก�I��� o>�?R�����E�f� luteal activity ����E>��Gc�ก���E���I?@G G� �� ��� 1 ����,��� 1 @G ��G�����EAQ�AQ�A> R��E�@E�>R��E�����T����>�K >�����A> �GA�EC�E��� (Means for Progesterone

metabolites quantity of the females evaluated )

NY�>@���? (Name of individuals)

NK� EGY>� (Month)

����� Progesterone metabolites �D��� (N) Mean ± SD (µg/g of feces)

�GA� EC�E��� (A���) �.�. �.�. (�� 52) 109 0.16 ± 0.1

�GA� EC�E��� (@����) �.�. �.�. (�� 52) 92 0.18 ± 0.1

Total / mean 201 0.17±0.1

ก���ก�T�NK� EGY>�ก��BC��P?_� E���� 2553

• �GA�EC�E���NY�> �A����

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�กก�������G��G��A> o>�?R��R��E�@E�>R�� (Progesterone metabolites) T����>�K >����� ��S �KNK� EGY>�ก��BC��P? _� E���� 2553 C��K �GA�NY�> �A���� ����G�����EAQ�AQ�o>�?R��R��E�@E�>R��E����� (Mean ± SD) E�Kก�� 2.53 ± 1.9 µg/g of dry feces

• �GA�EC�E���NY�> �@����� �กก�������G��G��A> o>�?R��R��E�@E�>R�� (Progesterone metabolites) T����>�K >����� ��S �K

NK� EGY>�ก��BC��P? _� E���� 2553 C��K �GA�NY�> �@����� ����G�����EAQ�AQ�o>�?R��R��E�@E�>R��E����� (Mean ± SD) E�Kก�� 2.49 ± 1.1 µg/g of dry feces

W�� �กก���ก�T��GA�EC�E�����S @> ��� C��K��G�����EAQ�AQ�A> o>�?R��R��E�@E�>R�����������G]GQ���KTก�QE��� ก�� ]�KC�����ก�K >�K �����@D��U� @_��� �K��G�����EAQ�AQ�o>�?R��R��E�@E�>R��RG�E����� (Mean ± SD) A> �GA����NY�>�A���� @J ก�K �@����� E�eก�Q>� [(P>0.05 , n=16 (ANOVA F=0.002, P = 0.962)] RG����KE����� (Mean ± SD) ���C�E�Kก�� 2.51 ± 1.5 µg/g of dry feces (C�@�� 0.56-6.0) ����E>��Gc�ก���E���I?@G G� �� ��� 2

����,��� 2 @G ��G�����EAQ�AQ�A> o>�?R��R��E�@E�>R��E�����T����>�K >�����A> �GA�EC�E��� (Means for

Progesterone metabolites quantity of the females evaluated)

NY�>@���? (Name of individuals)

NK� EGY>� (Month)

����� Progesterone metabolites �D��� ( N) Mean ± SD (µg/g of feces)

�GA� EC�E��� (A���) ก.C. �� 53 4 2.82 ��.�. �� 53 3 2.27 E�.�. �� 53 1 2.02 8 2.53 ± 1.9

�GA� EC�E��� (@����) ก.C. �� 53 3 2.25 ��.�. �� 53 4 2.87 E�.�. �� 53 1 1.67 8 2.49 ± 1.1

Total / mean 16 2.51 ± 1.5

��S ��SE�Y�>C�����กBC��� 1-2 C��K��>GNK� ����/2-3 EGY>���S �K��������EAQ�AQ�A> o>�?R��R��E�@E�>

R���K>�AQ �������ก���c�� (variable)>�K �K>E�Y�> ���� base line �K>�AQ @J @G _� B�������ก�E��Y�>�]I�A> o>�?R��E�f� luteal phase >�K N�GE�� RG�NK� ก�E�������� ��G��o>�?R��R��E�@E�>R��A> �GA�EC�E�����S @> �������ก�����Q���� ก���ก �>ก�ก��S�� C��K��G��o>�?R�����@G _� B�����E�f� luteal phase ��>�JKT�NK� 1- 6 µg/g W�� ����R�Q��Kก�E�������� ���E�f�� �>���S��E�f�]�T���ก������ก������E���K>E�Y�> ���� ���ก>�ก��T�NK� ����E��G� ก�K���S (ก��BC��P?_� E���� 2553) C�CX��ก���ก�����JKc@�C��P�?ก��EC�cJQT�EC�E�����S @> ���>�K �K>E�Y�> �K��GQ��

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��)��� 1 ก�p@G ���E��Y�>�]I�A> ��G��o>�?R��R��E�@E�>R�� (Progesterone metabolites) T��GA����NY�> �A������S �K NK� EGY>��ก��� 2552 _� E���� 2553

��)��� 2 ก�p@G ���E��Y�>�]I�A> ��G��o>�?R��R��E�@E�>R�� (Progesterone metabolites) T��GA����NY�> �@����� ��S �K NK� EGY>��ก��� 2552 _� E���� 2553

4. ��7#12$0�'#��5_2 �กก�������G��ก�E�������� ��G�����EAQ�AQ�A> o>�?R��R��E�@E�>R�� ���@G _� @B��ก��D �A> �K>�o>�?R��EC� (Gonadal activity) RG���P�ก���ก���]�K�Q> ����� ������@���? C��K��>GNK� �� 2552 (�ก���_� ����� 2552) �GA�EC�E�����S @> ���A> @��@���?E�FGEAEA��� ����G�����EAQ�AQ�A> o>�?R��R��E�@E�>R��>�JKT���G�����Tก�QE��� ก�� W�� ]�K�����@D��U� @_��� [(P> 0.05 , n = 201 (ANOVA F=1.216 , P = 0.272)] >�ก��S ����R�Q����E�f�]�T�

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���� EG���ก���Y>����������EAQ�AQ�A> o>�?R����D ���KE����� (Mean ± SD) A> ��G��o>�?R��R��E�@E�>R����>GNK� ��G� ก�K�E�Kก�� 0.17 ± 0.1 µg/g of dry feces (�KC�@��0.03-0.62) >��@G _� ��R�Q����]�K��ก�E�������� E�f�� �>� (non cyclic) I�Y>E�f�NK� ���]�K��ก�E�������� A> �� ]AK (ovarian inactivity) >�ก��S @G _� B�����]�K]GQ��ก���S �Q> T�NK� ����E��G� ก�K� ENK�EG���ก��ก��ก���ก�กK>�I�Q��SRG�>����ก ����� (2552) @>G��Q> ก��ก���� �A> Janine et al. (2002) ����� �_� ก���ก�T��GA��D��� 13 ���A> >E���กEI�Y> �Q�C��K�GA��D���ก�K���� I��� (6 �ก 13 ���) @G _� @B�����]�K��ก�E�������� A> �� ]AK (ovarian activity) G� BC��� 3

��)��� 3 ก�p@G ���E��Y�>�]I�A> �����o>�?R��R��E�@E�>R��T��GA�EC�E���E����� 6 ��� ����E��ก�������G�� 13-14 EGY>� (�กก�� �A> Janine et al,2002)

��S ��S�ก�� �A> Janine ����� (2002) C��K�GA����>�JKT�@K��@G >�ก 7 �����S�@G _� B�����E�f� luteal activity G� BC��� 4

��)��� 4 ก�p@G ก�E�������� A> �����o>�?R��R��E�@E�>R�����@G _� B�����E�f� luteal activity (�ก�� �A> Janine et al, (2002))

E�Y�>�Dก�C����c�ก���ก�T�NK� EGY>�ก��BC��P?_� E���� 2553 C��K�GA�EC�E�����S @> ���A> @��@���?E�FGEAEA������KE����� base line A> ��G�����EAQ�AQ�A> o>�?R��R��E�@E�>R��@J A�S� RG����KE����� (Mean ± SD) E�Kก�� 2.51 ± 1.5 µg/g of dry feces (C�@�� 0.56-6.00) W�� ��G�����EAQ�AQ�A> o>�?R����I�K �GA���S @> �����S����ก������E�f�]�T����� EG���ก���Y>��ก�@G B�����E�f� luteal phase RG�����R�Q��Kก�E�������� ���E�f�� �>���S��E�f�]�T���ก���

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���ก������E���K>E�Y�> ���� �K� �>����C��K>�AQ �������c��ก���Y>��NK� A> � �>��� 4 _� 10 @��GI? @>G��Q> ก���� �A> Schwarzenberger et al.(1998) W�� ]�K@��_����NK� �����������K�>�A> � �>�]GQE�Y�> �ก���_��A> �D������>�K ]�KEC�� C>��S ��SE�Y�>C����_� ���EAQ�AQ�A> R��E�@E�>R��A> �GA���S @> ���A> @��@���?E�FGEAEA������>�JKT�B�����E�f� luteal phase E�YS> �Q�C��K�����EAQ�AQ�>�JKT�NK� 1-6 µg/g W�� �ก�K �ก�� �A> Janine et al.(2002) ��������K>�JKT�NK� 3-24 µg/g RG�� �>�ก�E�������� A> o>�?R�����K E�f��� �shot cycle����long cycle� ���ก��E���� 32.8± 1.2 ��� �� 70.1± 1.6 ������DG��ENK�EG���ก��ก���� �A> Patton et al.(1999) ���C��K�GA�EC�E������>�JKT�@K��@G ��ก�E�������� A> � �>���S @> ���Y> �shot� ���long� �>ก�ก��S�� C���ก�ก��?_� ก�������GA�EC�E�����S @> ���A> @��@���?v ���>�JK�K��ก��B�T�Q@BC�G�Q>�EG���ก������R�Q�ก�E�������� A> � �>����E�f�]�T��J���EG���ก�� (synchronously) @>G��Q> ก���� �A> Janine et al.(2002) ����� �_� ก�C��K�GA�EC�E���@> ������>�JK>���T�ก� EC�E��S� EG���ก��@��_�������� �>�ก�E�������� A> o>�?R����Q���� ก��]GQ

RG�����Q��ก��G�����EAQ�AQ�A> o>�?R��R��E�@E�>R��T��GA�EC�E������EC���A�S��กT�NK� EGY>�ก��BC��P?_� E���� 2553 C����@>G��Q> ��E�f�]�T����� EG���ก��ก��ก�@� Eก�C�CX��ก���ก�����JKc@�C��P�?ก��>�K �K>E�Y�> W�� T�E�YS> �Q��� ]�K@��_@���]GQ�K�GA�EC�E�����S @> ���Eก�Gก���S �Q> I�Y>]�K EC�� �KC�ก�EC���A�S�A> �����o>�?R�����@G _� B�����E�f� luteal phase E�f����S �กT��>�EกY>� 2 ��E�K��S� ��S ��S�ก�� �ก���ก�T��GGD (Black Rhinoceros) ���Tก�QE��� ก���GA��ก���@�GC��KI�� �ก�����ก�c@�C��P�?��G�����EAQ�AQ�A> o>�?R��R��E�@E�>R����EC�����G��@J A�S�>�K �K>E�Y�> ��� >�JKT���G��@J (�กก�K 20 µg/g )��_� NK� กK>���>G ��S ��SAQ>�J����]GQT�ก���ก����S ��S�� E�f�EC�� @K��I��� A> c�ก���ก�E�K��S� ����S��� �DE�f����Q> GDE���ก�Eกe�AQ>�J����Dก���ก�������_� ก���ก������o>�?R��T�EC�cJQ (Testosterone) ��CX��ก���@���?EC���E���>�K �K>E�Y�> �K>]�.

5. 6/0%��01�$ @DI����GA�I�Y>@���?>Y��V RG�����]����>�JKT�@BCA> ก�EC�E��S� T�@_����กD��G �rUI�����ก��Eก�GA�S��Y>

���@��P�BCA> ก�A��C��P�?��D R��B��I�Y>���E�e��=����>���ก���@J W�� �rUIEI�K��SE�f�E�Y�> ���@��_����JQก��]GQ����]� �rUI@K��I��� Eก����AQ> ก�����@���?E> T�E�Y�> A> ก�AG���@��_T�ก��������TIQEAQก��@BC�G�Q>���@BC� @� ��@���? ��@�� ��SE> E�f�>�กEI��c�I��� ��������ก����E���_� B�����E����G (Stress) W�� _Y>�K�����@D��U�กก���GA���@���?>Y�����>�JKT�@BCA> ก�EC�E��S� (Janine et al., 2002 >Q _� Foose and Reece , 1997) ����S�ก���ก�T�R>ก@�K>]��� ������Q> GDE���ก�������G�����o>�?R�����E�f� fecal corticoid ENK� cortisol E�� ����������ก���ก�_� �r����>Y��V �����c�ก�����K>@�ABC��ก�@Y�C��P�?@���?���ก>�GQ�� E�f��Q�.

6. %0ก���0/�,0', >����ก ก> C�I�, N���� �? �r��� , @��P���ก��? ������@�, �F��>� �? �> �C��� ������ ECN�@�ก�. 2552. ก�������G

��G��o>�?R��R��E�@E�>R�� �ก���>�K >����� A> �GA� GQ����P�E>e�]W�?>���JR�E>@EW ECY�>�D]�TNQT�ก����E���@B��ก��D �A> �� ]AK. �� �ก�����ก� . ������ <=�>����ก�?������� @��@���?E�FGEAEA��� �.N�����.

Erich Mostl, Sophi Rettenbacher and Rupert Palme. 2005. Measurement of Corticosterone Metabolites in Birds� Droppings: An Analytical Approach. Ann. N.Y. Acad. Sci. 1046: 17 34.

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Janine L. Brown, Astrid C. Bellem, Michael Fouraker, David E. Wildt and Terri L. Roth. 2001. Comparative Analysis of Gonadal and Adrenal Activity in the Black And White Rhinoceros in North America by Noninvasive Endocrine Monitoring. Zoo Biology. 20:483-486.

Janine L. Brown, Steinman,K. 2004. Endocrine manual for the reproductive assessment of domestic and non-domestic species, 2nd edition, USA : Smithsonian institution.

R. Plame, S. Rettenbacher, C. Touma, S. M.El-Bahr and E. Mostl. 2005. Stress Hormones in mammals and birds Comparative Aspects Regarding Metabolism , Excretion, and Noninvasive Measurement in Fecal Samples. Ann. N.Y. Acad. Sci. 1040: 162 171.

Rupert Palme. 2005. Measuring Fecal Steroids Guidelines for Practical. Ann. N.Y. Acad. Sci. 1046: 75 80

Rupert Palme. 2005. Measuring Fecal Steroids Guidelines for Practical Application, Academy of science, New York. Schwarzenberger F, Walzer C, Tomasova K, Vahala J, Meister J, Goodrowe KL, Zima J, Strauß G and Lynch M. 1998. Fecal

progesterone metabolite analysis for non-invasive monitoring of reproductive function in the white rhinoceros ( Ceratotherium simum ).Anim Reprod Sci 53:173-90.

Toni E. Ziegler, and Daniel J. Wittwer. 2005. Fecal Steroid Research in the Field and Laboratory Improved Methods for Storage, Transport, Processing, and Analysis. University of Wisconsin. American Journal of Primatology , 67 : 159-174.

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Endocrine monitoring of Testicular and Ovarian activity in the White Rhinoceros at Khao Kheow Open Zoo by non-invasive method.

�5P�,(� #QR�(, 1, �7�*'2�กUP� &����$�& 1, �'�5� %) ��7ก� 1, ก�)'� #;�')̀ 2 12$-7~�&�[ [��3V��7,%ก'5��' 2

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3�(�.5�0 �กก�������E���I?��G��o>�?R��EC�T�ก��K��GA�EC�cJQ��EC�E���A> @��@���?E�FGEAEA��� RG���P�ก���ก���]�K����� ���I�Y>��ก�����@���? GQ��ก�������E���I?�����o>�?R��E�@R�@E�>R�� (Testosterone) A> EC�cJQ ��o>�?R��R��E�@E�>R�� (Progesterone) A> EC�E��� �ก���>�K >����� �� Enzyme-linked immunosorbent Assay (ELISA) W�� �กก�������G��G�����EAQ�AQ�A> o>�?R��E�@R�@E�>R�����@G _� ก��D �A> ����@Y�C��P�?EC�cJQ (Testicular activity) T�NK� EGY>�ก�ก �� 2553 C��K �GA�EC�cJQ NY�> �>��Rก-W��� A> @��@���?E�FGEAEA�������G�����EAQ�AQ�A> o>�?R��E�@R�@E�>R�� E�������S I�G (Mean± SD) E�Kก�� 251.73 ± 94.37 ng/g, N=19 (C�@�� 94.82-442.05 ng/g) W�� _Y>�K����G�����EAQ�AQ��K>�AQ @J E�Y�>E����ก���� �ก���ก�>Y��V ���cK��

�>ก�ก��ST�ก�������G��ก�E�������� A> ��G��o>�?R��R��E�@E�>R�� ECY�>��ก� Ovarian activity T��GA�EC�E��� ��I�K NK� EGY>���_���� 2553 _� ก�ก �� 2553 C��K��G�����EAQ�AQ�A> o>�?R��R��E�@E�>R��E�������S I�G (Mean± SD) E�Kก�� 1.70±1.35 µg/g, N=23 (C�@�� 0.129-4.03 µg/g) W�� E�Y�>E�����E�����K�����o>�?R�����������G]GQก��ก���ก�กK>�I�Q��S��I�K NK� EGY>�ก��BC��P?_� E���� 2553 C��K���J�����Q���� ก�� RG��KE�����CYS�u� (average base line) A> �����o>�?R����I�K NK� EGY>���_����_� ก�ก �� 2553 ���KE�����CYS�u�o>�?R����Dก�KE�eก�Q>� W�� �K�����o>�?R�����������G]GQ�� � >�JKT���G�����@��_@G ]GQ_� B������� ]AK�� � ��ก��D �E�K��S� W�� o>�?R��R��E�@-E�>R�����������G]GQG� ก�K��� � >�JKT���G�����EAQ�AQ���D �� ]�K@��_@���]GQ_� ก���S �Q> ���ก>�ก��ก�C�CX��ก���� EC���I�K �GA�EC�cJQ���GA�EC�E���T�NK� �Q�EGY>�ก�ก �� 2553 �K��GQ�� ��S ��S�� ������DE�f����Q> GDE���ก�@�K�Eกe����>�K ECY�>�D�������E���I?��G��o>�?R��ECY�>C�@J��?����K>]�.

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(EC�E���) RG���P�ก���ก���]�K����� ���I�Y>��ก�����@���? GQ��ก�������E���I?��G�����EAQ�AQ�A> o>�?R��R��E�@-E�>R�� (Progesterone metabolites) T����>�K >����� (feces) A> �GA����>�JKT�@BCA> ก�EC�E��S� ����G@G ��I�K NK� EGY>��ก��� 2552 _� E���� 2553 ECY�>�G@>�_� ���E�f��ก��A> ก��D �A> �� ]AK�����E�f�]�]GQT�ก���S �Q> A> �GA�EC�E��� RG�E�YS> �Q�]�KC��K�GA���S @> �����ก���S �Q> T�NK� E��G� ก�K� �K��S ��SC��KT�NK� EGY>�ก��BC��P?_� E���� 2553 �GA�EC�E�����S @> ���E��������R�Q����@G _� ก��D �A> �� ]AK RG���ก�EC���A�S�A> ��G��o>�?R��R��E�@E�>R�����@G _� B�����E�f� luteal phase E�f����S �กT��>�EกY>� 2 �����cK�� (N���� �?�����, 2553) �>ก�ก��S�กก����C��K�GA�EC�cJQ��EC�E�����ก�c@�C��P�?ก��>�K �K>E�Y�> T�NK� �Q��� 2553 �� �� � E�f�����K@�T�@DI������E�f�]�]GQT�ก���S �Q> A> EC�E���>�JK ���ก>�ก��ก�GDE���ก���ก����cK�� A> >����ก����� (2552) ��A> N���� �?����� (2553) �� ]�KE����ก�������G��G�����EAQ�AQ�A> o>�?R��T�EC�cJQI�Y>E�@R�@E�>R���กK>� ����S� ������ <=�>����ก�?��������K��ก����@���ก��������@��? �I�������P�U���� �� ]GQ��G�Dก�����ก�T�E�Y�> ก�������G����G��A> o>�?R��EC�cJQ��EC�E���A> �GA�A�S�ECY�>��G��B��ก�E�������� A> �����o>�?R�����>�@G _� ���E�������� ���Eก�GA�S�B�T����@���?RG�E�C�o>�?R��R��E�@E�>R�� (Progesterone) ���E�f�o>�?R�����Eก����AQ> ก��ก��ก]AK����S �Q> T�EC�E��� ���_� o>�?R��EC�cJQI�Y>E�@R�@E�>R�� (Testosterone) ECY�>���R�N�?T�ก�TNQE�f�u�AQ>�J����@D��U@DI���ก���Gก�ก� E��S� �>�@�> �K>C��P�ก��A> @��@���?E�FGEAEA��� �K>ก�E�f��J��?ก� IK ก�>����ก�?����ก��r�����K V T�ก�EC����D������Nก�T�@BCA> ก�EC�E��S� >��E�f�ก�>����ก�?�>ก_���>������ ���I�กI��� N��BCT�>����K>]�. 1.1 -7.&7�,+&�58�ก��[\กU�

1. ECY�>���E���@BC� @�������A> ����@Y�C��P�?��ก�E���UC��P�?A> ���Nก��GA�T�@��@���?E�FGEAEA���

2. ECY�>��ก�ก�E�������� A> ��G��o>�?R��R��E�@E�>R����E�@R�@E�>R�� RG���P�ก�@ก�G�ก>����� 3. ECY�>TIQ���AQ>�J����E�f����R�N�?�K>ก�EC�A��C��P�?�GT�@��@���?E�FGEAEA���

1.2 (��&�V�(�:#�$�5 �����(�.���-$T./��3-�กก���'-�5 ก���ก����S ��SE�f�ก�C�@J��?���ก�E�������� A> ��G��o>�?R��T�@���?ECY�>TNQ���E���@B��ก��D �A>

����@Y�C��P�? >�ก��S �DTIQ���AQ>�J�CYS�u����@��_�D]������ก�?TNQ���R�N�?]GQI�กI�� �����S ก��D�TNQกQ]A�>��rUIEก����ก������@Y�C��P�?, @��@G�BC@���? (welfare), ก�>����ก�?���C�ก�� N��BC (Conservation) ECY�>ก�������� ก���Gก�ก� E��S� TIQ@���?��CX��ก���ก�A��C��P�?��P���N��]GQ

1.3 ก�031��('. �YUZ����8 /8�ก���'-�5

�กก��������� ��K V ��AQ>@�����u��Kก�E�������� A> ��G��o>�?R��EC� ก�Eก�G� �>�ก�E�f�@�G��@BC�G�Q>��Q���Q��K��c��K>ก�@G CX��ก���� EC�A> @���? T��r������TNQ��P�ก���ก�N������RG�]�K��ก�����@���?I�Y>�Dก������ ��� (Noninvasive methods) GQ��ก���ก�CX��ก�����������G@E����>�G?T����>�K �J�@���?����P�ก������ ELISA ��E�Y�>�K ����ก���A> ก�]กก��D���ก������I�K >���E����>����>G����Eก�GT�ก��G@>� ��G>�JKT�ก��K� A> competitive ELISA (Brown et al., 2004) W�� ���E�f���P����EI��@���E�f�E��Y�> �Y>@D��UT�ก�

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�D�TNQ���E���@B��� GQ�o>�?R����TNQ>P������E�f�]�A> ก�����ก��D �A> �����K>�]�Q�K>��@_�BCA> ก�@Y�C��P�? (adenocortical activity and reproductive status) ]GQ>�K �����@��P�BC

�>ก�ก��S�DG�����@D��UA�S�@J A> ก�������D���EAQT�@_�BCA> ก�@Y�C��P�?T�@���?���>�JKT�@BCA> ก�EC�E��S� ���r�����K V �����>��P�C��K>���@��J��?C��P�?��S��Y>ก��D���EAQT�_� o>�?R�� o>�?R���Y>�r����T�ก�A��E��Y�>�@JKก�����ก�@Y�C��P�?���J���A> ก�I��� o>�?R���K V @��_TNQE�f�G�N��N�S��G@B��@BCA> ก�@Y�C��P�?]GQ (Brown et al., 2002)

2. �'*�ก��[\กU� 2.1 07#ก�P� ( Materials Essential Component of the EIA )

• Solid Phase: polystyrene microtiter plate (Nunc Immuno plate) • Monoclonal Antibody: Pregnane CL-425 • Polyclonal Antibody: Testosterone R 156/7 • Coating buffer: Carbonate/bicarbonate buffer pH 9.6 • Wash solution: NaCl and Tween 20 solution • Enzyme conjugate (tracer): Pregnane & Testosterone Horseradish peroxidase (HRP) • Assay buffer: Phosphate buffers pH 7.0 • Standards or unlabeled antigen: Standards Pregnane & Testosterone (Sigma Diagnostics) • Substrate: ABTS and catalyst (Hydrogen peroxide) Substrate • Reading: Spectrophotometer or Plate reader

2.2 ���05��,����� (Animals sample)

• �GA�EC�cJQ �D��� 1 ��� NY�> �>��RกW��� >�� 8 �� • �GA�EC�E��� �D��� 1 ��� NY�> �A���� >�� 17 ��

2.3 �'*�ก�� (Methods) �Dก���ก�T��GA�EC�cJQ��EC�E��������CX��ก���ก�����JKc@�C��P�? �D��� 1 �JK A> @��@���?E�FGEAEA��� RG�Eกe����>�K >�������I�K NK� EGY>���_���� 2553 _� ก�ก �� 2553 RG���A�S��>���K�V G� ��S�Y> Eกe����>�K >������GA� (Collection feces): @�K�Eกe����>�K >�����A> �GA�EC�cJQ��EC�E����K����� (20-50 ก���) @��GI?�� 3-4 ���>�K RG�E�Y>กEกe��K���>�K >������������ก���E�f�E�YS>EG���ก����]�K��E����@G�>Y����>�JKT����>�K ECY�>TIQ]GQ@E����>�G?o>�?R�������ก�ก�������ก��>�K @��DE@�> �Dก�Eกe�E�C�T�NK� ENQECY�>�t> ก��o>�?R��_Jก�D���ก@ GG T@KT�_� W��C�@��ก����������@���?��������Eกe� �Dก���@JKIQ> �������ก���E���I?�����o>�?R�� ก�Eกe���ก����>�K I�Y>�DTIQIQ (Preservation with Freezing or Drying immediately): ก�������� ]�K@��_�D���>�K TIQIQ ]GQ��� @��_�Dก�Eกe���ก����>�K ]�QT�ก�K> ���@ �����<��GECY�>�t> ก��]�KTIQ���>�K @��c�@ก�����NYS�I�Y>@ �ก��S��D]�Eกe�]�QT��JQNKAe >��IBJ�� -20 > �EW�EW��@

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�D���>�K ���NKAe Eกe���ก�@BC��DTIQIQ GQ��ก�]�K���NYS�>>ก�ก���>�K GQ��ก��DEAQ�JQ>� (Hot air oven) E�f�����E��]�K�Q>�ก�K 96 N���R� ���>��IBJ�� 60 > �EW�EW��@ ���SDI��ก]�KE�������� >�ก �ก��S�Eกe���ก����>�K GQ��ก�T@KI��IK>���@ T@K@�GJG���NYS� �FG<TIQ@����D]�Eกe�]�QB�T��JQ������>��IBJ�� ���E�e����YG ECY�>�t> ก��ก�E�������� A> @E����>�G? (Erich et al., 2005; Tony et al., 2005) �Q��>�D]�@ก�Go>�?R���K>]� ก�@ก�G@E����>�G?o>�?R���ก�J�@���? (Fecal Extraction):

�Dก�@ก�GGQ����P�ก��Q� (Dry and Wet Weight Fecal Extraction Boiling Method) TNQ���>�K �J��กก��@�>GD 0.1-0.2 g @ก�GGQ�� Ethyl alcohol 90 % �K��ก��ก��ก��Q�GQ������Q>� 95 > �EW�EW��@T� Water bath EA�K���r��EI���� GQ��E��Y�> Vertex �� Centrifuge ���DG�� �Q�Eกe�@��������]GQ]�QT� dilution buffer NKAe ���>��IBJ�� -20 > �EW�EW��@ ECY�>�>ก���E���I?�K>]� ก���E���I?�����o>�?R�� (Assay Protocols): �Dก���E���I?�����o>�?R��R��E�@E�>R�� (Fecal Progesterone) ��o>�?R��E�@R�@E�>R�� (Fecal Testosterone) GQ����P� Enzyme immunoassay �� Competitive ELISA (Enzyme Linked Immuno Sorbent Assay) ก���E���I?AQ>�J� (Data analysis): Eกe�AQ>�J��KE�������G��o>�?R���K V ���������G]GQ��G�DE�f��KE�����u�A> o>�?R�� (Average baseline) E�����E������G��o>�?R�����������G]GQE����ก����G��o>�?R��RG�E�����T�ก���ก�A> N���� �? �����(/2553) ��>GNK� �� 2552 ��ก��BC��P?_� E���� 2553 ��E���I?AQ>�J����]GQGQ����P�ก�� @_����� ANOVA E�����E��������ก�K ��I�K �JKA> I�K��ก��G�> RG���P� LSD �� E�����E��������ก�K ��I�K ก��K�RG���P� Duncan�s new Multiple Range test �����G�����ENY�>���� 95 %

3. _2ก��[\กU� 12$0�'#��5_2

�กก�������G����G��o>�?R��T��GA�EC�cJQ��EC�E���A> @��@���?E�FGEAEA��� RG���P�ก���ก���]�K����� ������@���? GQ��ก�������E���I?��G�����EAQ�AQ�A> o>�?R��E�@R�@E�>R�� (Fecal Testosterone) ��R��E�@E�>R�� (Fecal Progesterone) ���DG�� T����>�K >����� (feces) A> �GA����>�JKT�@BCA> ก�EC�E��S� ����G@G ��S �KEGY>���_����_� ก�ก �� 2553 ]GQc��D�ก���K��I��AQ>����� �K>]���S ก��[\กU� Testicular activity 8�1�.6��%)[_9/ �กก���ก�ก��D �A> ����@Y�C��P�?EC�cJQ GQ��ก�������G��G��o>�?R��E�@R�@E�>R�� �ก>�����T�NK� EGY>�ก�ก �� 2553 C��K �GA�EC�cJQ NY�> �>��RกW��� ����G�����EAQ�AQ�A> o>�?R��E�@-R�@E�>R��T�NK� EGY>�G� ก�K�E�������S I�G (Mean± SD) E�Kก�� 251.73 ± 94.37 ng/g, N=19 (C�@�� 94.82-442.05 ng/g) W�� E�C�T�NK� EGY>�ก�ก �� 2553 C��K��G�����EAQ�AQ�A> o>�?R��� >�JKT���G��@J �K>E�Y�> �K��ก����CX��ก���c@�C��P�?ก��EC�E�����CX��ก���� EC�>Y��V ���ก>�GQ�� c�ก���E���I?o>�?R��@��_@G G� BC��� 1

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��)��� 1 ก�p@G ���E��Y�>�]I�A> ��G��o>�?R��E�@R�@E�>R�� (Fecal Testosterone) T��GA�EC�cJQ NK� EGY>�ก�ก �� 2553

��S ��SC��K��G��o>�?R��E�@R�@E�>R��E�����T��GA�EC�cJQA> @��@���?E�FGEAEA���>�JKT���G����������EAQ�AQ�

@J �ก RG�C�@��A> ���EAQ�AQ�o>�?R��E�@R�@E�>R�����������G]GQ�ก�K �ก�� �A> Brown ����� (2002) ��������K>�JKT�NK� 2-200 ng/g >�ก��S ���KE�������S I�G (Overall mean) ��� 16.8±3.1 ng/g �>ก�ก��S�� ����_� ก�����GA����GGDEC�cJQT�@I��u>E���ก]�K@G _� ก�EC���A�S�I�Y>�G� E�f�NK� XGJก�>�กGQ�� (Brown et al., 2002)

ก��[\กU� Ovarian activity 8�1�.6��%)[%&�5

�กก���ก�ก��D �A> >�����@Y�C��P�?EC�E��� GQ��ก�������G��ก�E�������� A> ��G��o>�?R��R��E�@E�>R�� T����>�K >�����A> �GA�EC�E��� NY�>�A���� ��I�K NK� EGY>���_����_� ก�ก �� 2553 C��K �GA�EC�E���G� ก�K� ����G�����EAQ�AQ�A> o>�?R��R��E�@E�>R�� E�������S I�G (Mean±SD) E�Kก�� 1.70±1.35 µg/g, N=23 (C�@�� 0.129-4.03 µg/g) ��S ��SC��K��G��o>�?R���K>�AQ �������ก���c�� (variable) >�K �K>E�Y�> @G _� B�������ก�E��Y�>�]I�A> o>�?R�� I�Y>ก��D �A> �� ]AK ��S ��S��I�K NK� EGY>���_����_� ก�ก �� 2553 ���KE�����CYS�u� (Average baseline) A> o>�?R��R��E�@E�>R��G� ก�K�E�Kก�� 1.26±1.02 µg/g �>ก�ก��S�� C�CX��ก���ก�c@�C��P�?�K��GQ�� ����E>��Gc�ก���E���I?@G G� BC��� 2

��)��� 2 ก�p@G ���E��Y�>�]I�A> ��G��o>�?R��E�@E�>R�� (Fecal Progesterone) T��GA�EC�E��� NK� EGY>���_���� _� ก�ก �� 2553

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M

M

E�Y�>E�����E�����K�����o>�?R�����������G]GQก��ก���ก�กK>�I�Q��SA> N���� �?����� (2553) ��I�K NK� EGY>�ก��BC��P?_� E���� 2553 C��K���J�����Q���� ก�� RG���������EAQ�AQ�A> o>�?R��R��E�@E�>R��T�NK� EGY>���_����_� ก�ก �� �� � ����R�Q�����K>E�Y�> �กNK� EGY>�กK>�I�Q��S @G G� BC��� 3 ��S ��SE�Y�>E�����E�����KE�����CYS�u�A> �����o>�?R����I�K NK� EGY>���_����_� ก�ก �� 2553 (1.26±1.02 µg/g) ก��NK� EGY>�ก��BC��P�? 2553 _� ก�ก �� 2553 (2.53±1.93 µg/g) C��KNK� EGY>���_����_� ก�ก �� 2553 ���KE�����CYS�u�o>�?R����Dก�K �K���KE�����CYS�u�T�NK� EGY>�G� ก�K�Tก�QE��� ก��ก��ก���ก�A> Brown �����(2002) ��������K �KE�����CYS�u� (Average baseline ) ��S I�GA> o>�?R��R��E�@E�>R�����@ก�G�ก>������GA� �D��� 13 ������KE�Kก�� 1.22±0.41 µg/g W�� T��D�����S 3 �����ก�����JKc@�C��P�?ก��EC�cJQ �K]�K]GQ�Jก (Brown et al.,2002)

�>ก�ก��SE�Y�>C�����กBC��� 4 C��K����ก���Tก�QE��� ก��ก���ก�A> Brown ����� (2002) ���C��KEC�E���� ������>�JKT�@BCA> ก���G@G ����NK� ����� ]AK]�K@G _� ก��D � (Ovarian inactivity) �ก��S���@G _� NK� �����ก�E�������� E�f�� �>���@�S�����@���ก��]� (@G G� BC��� 4)

��)��� 3 ก�p@G ���E��Y�>�]I�A> ��G��o>�?R��R��E�@E�>R�� (Fecal Progesterone) T��GA����NY�> �A���� ��S �K NK� EGY>��ก��� 2552 _� ก�ก �� 2553

* M �Y>NK� ���E������ก�c@�C��P�?

��)��� 4 ก�p@G ���>�K ���E��Y�>�]I�A> ��G��o>�?R��R��E�@E�>R�� (Fecal Progesterone) T��GA����>�JKT�@K����G@G A> @I��u>E���ก �กก���ก�RG� Brown ����� (2002)

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4. ��7# �กc�ก�������E���I?��G��o>�?R��E�@R�@E�>R�� (Fecal Testosterone) T��GA�EC�cJQ��o>�?R��R��E�@-

E�>R�� (Fecal Progesterone) T��GA�EC�E���A> @��@���?E�FGEAEA��� ��I�K EGY>���_����_� ก�ก �� 2553 C��KEC�cJQ����G��o>�?R��E�@R�@E�>R��E�������S I�G (Mean± SD) E�Kก�� 251.73±94.37 ng/g W�� ����G������K>�AQ @J @K��T�EC�E������KE�����CYS�u� (Average baseline) A> �����o>�?R��R��E�@E�>R�� E�Kก�� 1.26±1.02 µg/g (C�@�� 0.13-4.03 µg/g) W�� ��Dก�KNK� EGY>�ก��BC��P? _� E���� 2553 E�eก�Q>� �>ก�ก��S�� C�CX��ก���ก�c@�C��P�?ก��EC�cJQT�NK� �Q�EGY>�ก�ก �� 2553 �K��GQ��

5. 6/0%��01�$ @DI���ก���ก����S ��SE�f�ก�@�K�������G��ก�E�������� A> ��G��o>�?R��R��E�@E�>R�� ������@>�

o>�?R��E�@R�@E�>R��T�NK� ����E�������ก�Eกe����>�K EC�� NK� @�S�V E�K��S� �DTIQc�ก���E���I?� @K�����D������>�K �Q>� RG�E�C�ก�������E���I?��G�����EAQ�AQ�A> o>�?R��E�@R�@E�>R��W�� �����ก�����T�NK� EGY>��K>V ]�ECY�>@��_�D�TNQT�ก����E����K baseline A> �����o>�?R��]GQ ��S ��S�����ก���ก�E�����E����ก��ก��EC�cJQ���>Y��V �K��GQ�� �>ก�ก��ST�ก���A> �GA�EC�E����K�����o>�?R�����������G]GQ�� � >�JKT���G�����@��_@G ]GQ_� B������� ]AK�� � ��ก��D �E�K��S� �� ]�K@��_@���]GQ�K��ก���S �Q> I�Y>]�KE�Y�> �ก�� � C�CX��ก���ก�c@�C��P�?T�NK� �Q�EGY>�ก�ก �� 2553 W�� o>�?R��R��E�@E�>R�����������G]GQG� ก�K��� � >�JKT���G����D �� ������DE�f����Q> GDE���ก�@�K�Eกe����>�K ECY�>�D�������E���I?��G��o>�?R��ECY�>C�@J��?����K>]�.

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N���� �? �r��� , @��P���ก��? ������@�, ���� ECN�@�ก� ����UN ����{�. 2553 .ก����E���@B��ก��D �A> �K>�o>�?R��EC�T�ก��K��GA�EC�E���A> @��@���?E�FGEAEA��� RG���P�ก���ก���]�K����� ���I�Y>��ก�����@���? GQ��ก�������E���I?�����o>�?R��R��E�@E�>R��.�� �ก�����ก�. ������ <=�>����ก�?������� @��@���?E�FGEAEA��� �.N�����.

Brown, J., Walker, S. and Steinman, K. 2004. Endocrine manual for the reproductive assessment of domestic and non-domestic species, 2nd edition, USA : Smithsonian institution.

Erich Mostl, Sophi Rettenbacher and Rupert Palme. 2005. Measurement of Corticosterone Metabolites in Birds� Droppings: An Analytical Approach. Ann. N.Y. Acad. Sci. 1046: 17 34.

Rupert Palme. 2005. Measuring Fecal Steroids Guidelines for Practical. Ann. N.Y. Acad. Sci. 1046: 75 80

Rupert Palme. 2005. Measuring Fecal Steroids Guidelines for Practical Application, Academy of science, New York. Toni E. Ziegler, and Daniel J. Wittwer. 2005. Fecal Steroid Research in the Field and Laboratory Improved Methods for

Storage, Transport, Processing, and Analysis. University of Wisconsin. American Journal of Primatology , 67 : 159-174.

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Assessment of Gonadal and Adrenal activity in non-breeding female Black necked stork by non-invasive endocrine monitoring.

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�กก����E���@B��ก��D �A> �K>�o>�?R��EC���>�G����� (Gonadal and Adrenal Activity) T��กก��@�>GDEC�E������]�K����JKRG�ก�������G��ก�E�������� A> ��G��o>�?R�� W�� E�f�ก���ก����Eก����AQ> ก��ก�������G�����A> E@����>�G?o>�?R�����E�f�o>�?R��EC� (RG�E�C� fecal Progesterone) ��o>�?R�����E����G �ก�K>�]�Q�K>��I�K NK� EGY>�CX�B�� 2553 _� ก�ก �� 2553 RG�c�ก���ก�C��K �กก��@�>GDEC�E������]�K����JK���KE�����u������o>�?R�� Progesterone (fecal Progesterone) E�Kก�� 0.19±0.06 ng/g of dry feces (C�@�� 0.03-0.28 ng/g) ��E�Y�>E�����E����ก��AQ>�J�ก���ก�T��กก��@�>GDEC�E����������JK�Q���I�K NK� �� ������ 2551-2552 C��K�กก��@�>GD���]�K]GQ����JKT��r�������������o>�?R��G� ก�K�@J ก�K�กก��@�>GD�������JK�Q� (���KE�����u������o>�?R�� E�Kก��0.11± 0.03 ng/g) >�K �����@D��U� @_��� (P< 0.05) >��@��_>����]GQ�K �K>�o>�?R��EC�A> �กก��@�>GD���]�K]GQ����JK�� � ��ก��D �>�JK (active gonadal) @DI���ก���E���I?@B��ก��D �A> �K>�]�Q�K>I�Y> Adrenal GQ��ก�������Go>�?R�� Cortisol metabolites C��K �กก��@�>GDEC�E������]�K]GQ����JKT��r����������G�����EAQ�AQ�A> o>�?R�� Cortisol metabolites E�����E�Kก�� 15.15±6.44 ng/g of dry feces (C�@�� 3.56 26.34 ng/g) W�� ก���ก����S ��S]GQ�G@>�ก�I��� o>�?R��T�ก��K� Corticoids (ENK� o>�?R�����E����G) A> �K>�]�Q�K> GQ��ก��G�> TIQ��ก�E�������� ��G��A> ACTH �Q��Dก�������G�K�����o>�?R�� Cortisol metabolites E�����E����กK>���I�� ก��G�> TNQ Adrenocorticotrophic hormone (ACTH) T��กก��@�>GDEC�E��� W�� C��K ����G��o>�?R�� Cortisol metabolites E�����E�Kก�� 14.30± 4.14 �� 28.32 ± 13.60 ng/g of dry feces ���DG�� ��S ��S�กก��@�>GD���]�K]GQ����JK�������o>�?R��E�����Tก�QE��� ก���กก��@�>GDEC�E�������]�กK>��������ก���G ACTH (�ก�K >�K ]�K�����@D��U� @_��� P>0.05) �KE�Y�>E�����E����ก��B�I�� �����ก���GACTH C��K���������o>�?R����Dก�K>�K �����@D��U� @_��� (P<0.05) �� @��_ก�K�]GQ�K �กก��@�>GD���]�K����JKT��r���������G�����EAQ�AQ�A> o>�?R�����E����G>�JKT���G�����]�K@J c�G�ก�� �>ก�ก��S�� @��_@G _� ก��D �A> �K>�]�Q�K>����K��E�f��ก��>�กGQ��. (V��V�(�: : �กก��@�>GD; Progesterone metabolites; Cortisol metabolites; Adrenocorticotrophic hormone

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ABSTRACT The assessment of Gonadal and Adrenal activity in non-breeding females Black necked stork by non-invasive endocrine monitoring, with respect to evaluating of fecal reproductive steroid metabolites (especially fecal progesterone) and adrenal corticoids level were characterized between May to July 2010. The non-breeding females of captive Black necked stork group found that overall baseline concentrations of fecal progesterone were averaged 0.19±0.06 ng/g (range 0.03-0.28 ng/g). When compared with the paired female during the study in 2008 to 2010, there was significant different in average baseline concentration among present non-paired females and paired female birds group (P< 0.05, n=121). Before that from paired female, mean average baseline concentrations of fecal progesterone is equal to 0.11± 0.03 ng/g. This indicated that all non-paired females showed evidence at least some gonadal activity on the basis of fecal progesterone analysis. The assessment of Adrenal activity with evaluating of Cortisol metabolites, overall baseline concentrations of cortisol metabolites were averaged 15.15±6.44 ng/g (range 3.56 26.34 ng/g). This assay was use experiment to stimulate adrenocortical activity (i.e., increase circulating GC levels) is the so-called ACTH challenge test. In this respect, the most widely was determined before and after an adrenocorticotropin hormone (ACTH) by using the cortisol assays, cortisol metabolites was detected in fecal dilutions of females Black necked stork. The result has been found that overall concentrations of fecal cortisol metabolites before and after an adrenocorticotropin hormone (ACTH) were averaged 14.30± 4.14 and 28.32 ± 13.60 ng/g of dry feces, respectively. A one-way ANOVA showed non-significant different in average cortisol metabolites level between non-paired females and paired females birds group during the study before ACTH injection. But there was significant different in average fecal cortisol metabolites level (P < 0.05), when compared with after ACTH injection. In addition, it suggested that non-paired females had stress hormone in usual level, the study also found that all most non-paired females showed evidence of adrenal activity on the basis of fecal cortisol metabolites analysis.. Keywords: Black necked stork; Progesterone metabolites; Cortisol metabolites; Adrenocorticotrophic hormone

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W�� A�S��>�A> ก���ก����]GQGDE���ก�T�ก���ก����S ��S��S�Eก����AQ> ก��ก�E�����E���������o>�?R��EC����_JกA��_K�>>ก� (gonadal steroid metabolites) ��o>�?R�����E����G���E�f�o>�?R��T�ก��K� GCs (glucocorticoid) ���TNQE�f�G�N��N�S��Gก��DI�Q���A> �K>�]�Q�K> (Adrenal) T��กก��@�>GD EC�E������]�K����JKA> @��@���?E�FGEAEA��� RG�TNQก���E���I?���E�������� A> ��G��o>�?R����]�K�Dก������ ���I�Y>��ก�����@���? (�กE�Q�A�S��>�A> ก���G ACTH) ��S ��Sก�����ก������ immunoassay ���E�f�ก������ @����������Q> �D�C�@J��?����KE����� ก���E���I?���TNQ��S������@��P�BCT�ก�������G��G��A> >��C��P?@E����>�G?�K V T����>�K >�����]GQG��K]I� T�ก���A> o>�?R��T�ก��K� GCs ��P�����D�E�����E����c��������TNQก��>�K ก�Q A� �Y>ก�TNQ Adrenocorticotrophic hormone (ACTH) �D��G@>�ก�E�������� ��ก�������ก�D�TNQGQ��ก�ก����Q��K>�I��ก]� (Adrenal cortex) T�@���?ECY�>E�����E����c�A> @���?�K����� ��S ��S@��_ก���D]GQGQ��ก���G ACTH EAQ]��Q�������G��������_JกI��� >>ก�E�����E����ก�� � ���S ก�����ก���E���I?� @�������TIQ_Jก�Q> �K��DGJEI�Y>���ก���D]GQ�D�ก (ENK�T�@���?���Tก�Q@JUC��P�?) T�@BCก��?���@���?�Q> >�JKT�@BCA> ก�>�JKT�CYS�����Dก�G ก���ก�� N������GQ��ก�����EI�K��S�� ������DE�f��Q> GDE���ก�>�K ��� GQ����P�ก���ก���]�K��ก�����@���? I�Y> noninvasive method W�� �����EI��@�ก��@���?���>�JKT�@_�@BCG� ก�K�

(Palme, 2005).

2. �'*�.V�%�'�ก���'-�5 2.1 07#ก�P� ( Materials Essential Component of the EIA )

• Solid Phase: polystyrene microtiter plate (Nunc Immuno plate) • Monoclonal Antibody: Progesterone CL-425 • Polyclonal Antibody: Cortisol R 4866 • Coating buffer: Carbonate/bicarbonate buffer pH 9.6 • Wash solution: NaCl and Tween 20 solution • Enzyme conjugate (tracer): Progesterone & Cortisol Horseradish peroxidase (HRP) • Assay buffer: Phosphate buffers pH 7.0 • Standards or unlabeled antigen: Standards Progesterone & Cortisol (Sigma Diagnostics) • Substrate: ABTS and catalyst (Hydrogen peroxide) Substrate • Reading: Spectrophotometer or Plate reader

2.2 ���05��,����� (Animals sample)

• �กก��@�>GDEC�E��� ����� ]�K]GQ����JK A> @��@���?E�FGEAEA��� �D��� 2 ��� 2.3 �'*�ก�� (Methods) %กi3���05��,&92�กก�$��(0.V� (Collection dropping) @�K�Eกe����>�K �J�A> �กก��@�>GDEC�E������]�K]GQ����JK @��GI?�� 1-3 ���>�K (NK� EGY>�CX�B��_� ก�ก��� 2553) E�Y>กEกe��K���>�K �J��������ก���E�f�E�YS>EG���ก��ECY�>TIQ]GQ@E����>�G?o>�?R�������ก�ก�������ก��>�K @��DE@�>T�

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���>�K �J����Q> ����ก���@GTI�K ]�KIQ ��]�K��E����@G�>Y����>�JKT����>�K �J����]GQ���D]�������G��G��o>�?R��R��-E�@E�>R�����>�?��W>��K>]� (Toni, 2005) ก�$�7/�ก��%ก'.(��&%(��5. (Increasing stress level) ก����Q�TIQ�กEก�Gก�I��� o>�?R�� GCs T���G��@J GQ��ก���G ACTH I�Y>TNQ��P�GQ��ก��DTIQ�ก�กT�I�Y>>�JKT�@_�ก��?กGG��T�NK� E��I��� ECY�>TIQ��ก�I��� o>�?R���>�?��W>� �Q��Dก�Eกe����>�K �J�ECY�>�D�������G��G��o>�?R�����E����G���Eก�GNYS� E�����E����ก�����>�K �ก�����]�K]GQก����Q�I�Y>��G ACTH %กi3��กU����05��,+�`0�V�8+/1+/, (Preservation with Freezing or Drying immediately) �Dก�Eกe���ก����>�K ]�QT�ก�K> ���@ �����<��GECY�>�t> ก��]�KTIQ���>�K @��c�@ก�����NYS�I�Y>@ �ก��S��D]�Eกe�]�QT��JQNKAe >��IBJ�� -20 > �EW�EW��@ ECY�>Eกe���ก�@BCA> �J��ก�ก����E��� ��E�f�ก��t> ก��]�KTIQ@E����>�G?��ก�E�������� @BC]� �D���>�K ���NKAe Eกe���ก�@BC��DTIQIQ GQ��ก�]�K���NYS�>>ก�ก���>�K GQ��ก��DEAQ�JQ>� (Hot air oven) E�f�����E��]�K�Q>�ก�K 96 N���R� ���>��IBJ�� 60 > �EW�EW��@ ���SDI��ก]�KE�������� >�ก �ก��S��D]�@ก�Go>�?R���K>]� ก���ก�.�%��5�05.�j0���&�-�ก&92����� (Fecal Extraction)

�Dก�@ก�GGQ����P�ก��Q� (Dry and Wet Weight Fecal Extraction Boiling Method) TNQ���>�K �J��กก��@�>GD 0.1-0.2 g @ก�GGQ�� Ethyl alcohol 90 % �K��ก��ก��ก��Q�GQ������Q>� 95 > �EW�EW��@T� Water bath EA�K���r��EI���� GQ��E��Y�> Vertex �� Centrifuge ���DG�� �Q�Eกe�@��������]GQ]�QT� dilution buffer NKAe ���>��IBJ�� -20 > �EW�EW��@ ECY�>�>ก���E���I?�K>]� ก���'%(��$+�#�'&�Pj0���&� (Assay Protocols) �Dก���E���I?�����o>�?R��R��E�@E�>R�� (Progesterone metabolites) ��o>�?R���>�?��W>� (Cortisol metabolites) GQ����P� Enzyme immunoassay �� Competitive ELISA (Enzyme Linked Immuno Sorbent Assay) ก���'%(��$+�6/0&92 (Data analysis) Eกe�AQ>�J��KE�������G��o>�?R���K V ���������G]GQ (Average baseline) E�����E������G��o>�?R��T��กก��@�>GD���]�K����JKT��r������E����ก����G��o>�?R��RG�E�����T��กก��@�>GD���]�K����JK���������JKA��C��P�?]GQ��>GNK� �� 2551-2552 ��E���I?AQ>�J����]GQGQ����P�ก�� @_����� ANOVA E�����E��������ก�K ��I�K �JKA> I�K��ก��G�> RG���P� LSD �� E�����E��������ก�K ��I�K ก��K�RG���P� Duncan�s new Multiple Range test �����G�����ENY�>���� 95 %

3. _2ก���'-�512$0�'#��5_2 �กก���ก�@B��ก��D �A> �K>�o>�?R��EC� (gonadal activity) GQ��ก���G��G�����EAQ�AQ�A> o>�?R��R��E�@E�>R�� (Progesterone metabolites) ECY�>TNQT�ก����E���@BCก����@��J��?C��P�? ����ก�ก��D �A> �K>�>�G����� (adrenal activity) T��กก��@�>GDEC�E������]�K]GQ����JK RG�ก���G��G��o>�?R�����E����G (Cortisol metabolites) ECY�>���E���@BCA> ก���Gก�ก� E��S� ��I�K NK� EGY>�CX�B�� 2553 _� ก�ก �� 2553 ]GQc�G� �K>]���S ก��[\กU� Gonadal Activity

�กก�������G��G��A> o>�?R��R��E�@E�>R�� (Progesterone metabolites) T����>�K �J��กก��@�>GDEC�E������]�K����JK C��K ����G�����EAQ�AQ�A> o>�?R��R��E�@E�>R��E����� (average baseline) E�Kก�� 0.19 ± 0.06 µg/g of dry feces, (N=24) RG�����G�����EAQ�AQ�>�JK��I�K (C�@��) 0.03-0.28 µg/g ����E>��Gc�ก���E���I?@G G� �� ��� 1

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��)��� 1 ก�p@G ���E��Y�>�]I�A> ��G��o>�?R��R��E�@E�>R�� (Progesterone metabolites)

T��กก��@�>GDEC�E������]�K����JK ��S �K NK� EGY>�CX�B�� 2553 _� ก�ก �� 2553

��)��� 2 ก�p@G ���E��Y�>�]I�A> ��G��o>�?R��R��E�@E�>R�� (Progesterone metabolites) T��กก��@�>GDEC�E������]�K����JK������JK]GQ T��� ������ 2551-2552

��S ��SE�Y�>C����E�����E������������EAQ�AQ�A> o>�?R��R��E�@E�>R�� (Progesterone metabolites)��I�K �ก

ก��@�>GDEC�E������]�K]GQ����JKT��r������ (BC��� 1) E����ก���กก��@�>GD�������JK (Mean±SD = 0.11±0.03 µg/g , C�@�� 0.06-0.21) ��]�K]GQ����JK (Mean±SD = 0.10±0.03 µg/g , C�@�� 0.06-0.16) T��� ������ 2551 _� 2552 (BC��� 2) C��K�K��������EAQ�AQ�A> o>�?R��R��E�@E�>R��T��กก��@�>GD���]�K]GQ����JK (�� 53) �� base line �K>�AQ @J ก�K���C�T��� 51-52 G� �� ��� 1 ��E�Y�>�Dก���E���I?GQ����P�ก�� @_��� C��K�ก���]�K]GQ����JKT��r����������G��o>�?R��@J ก�KRG�@G ���

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�ก�K >�K �����@D��U� @_��� (ANOVA, F=45.30, P=0.000) W�� @G _� B������K>�o>�?R��EC��� � ��ก��D � (active) >�K N�GE��

����,��� 1 @G ��G�����EAQ�AQ�A> R��E�@E�>R��E�����T����>�K �J�A> �กก��@�>GD (Means for Progesterone metabolites quantity of the females evaluated )

N��G@���? (Name of individuals)

NK� EGY>� (Month)

�KE���������� Progesterone metabolites (µg/g of feces) EC�E���]�K����JK �� 53 EC�E���]�K����JK �� 51-52 EC�E�������JK �� 51-52

ก��@�>GD C.�. 0.20 ± 0.06 0.11 ± 0.03 0.12 ± 0.03 ก��@�>GD ��.�. 0.18 ± 0.07 0.11 ± 0.03 0.10 ± 0.03 ก��@�>GD ก.�. 0.20 ± 0.04 0.10 ± 0.03 0.12 ± 0.03

Total / mean 0.19±0.06 b(N=24) 0.10±0.03 a(N=48) 0.11±0.03 a(N=49)

I��EI�� a �� b @G ���EI�Y>�I�Y>����ก�K ��I�K ก��K�>�K �����@D��U� @_��� E�Y�>E�����E��������ก�K �KE�������I�K �JKA> I�K��ก��G�> RG���P� LSD �� Duncan�s �����G�����ENY�>���� 95 %

ก��[\กU� Adrenal Activity �กก�������G��G��A> o>�?R���>�?��W>� (Cortisol metabolites) T����>�K �J��กก��@�>GDEC�E������]�K����JK

T�NK� EGY>���_���� 2553 _� ก�ก �� 2553 C��K ����G�����EAQ�AQ�A> o>�?R���>�?��W>�E����� (Average baseline) E�Kก�� 15.15 ± 6.44 ng/g of dry feces (C�@�� 3.56-26.34 ng/g, n = 28) ��E�Y�>E�����E������G�����EAQ�AQ�A> �K��EGY>�T�NK� ����E��G� ก�K� C��K �K��EGY>�����G�����EAQ�AQ�A> o>�?R���>�?��W>�Tก�QE��� ก�� (�ก�K >�K ]�K�����@D��U� @_��� P>0.05) ����E>��Gc�ก���E���I?@G G� BC��� 3

��)��� 3 ก�p@G ���E��Y�>�]I�A> ��G��o>�?R���>�?��W>� (Cortisol metabolites) T��กก��@�>GDEC�E������]�K����JK T�NK� EGY>�CX�B�� 2553 _� ก�ก��� 2553

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��S ��SAQ>�J�T�NK� �����ก��G�> ��G ACTH T��กก��@�>GDEC�E�����S�@��_�K ก��K�AQ>�J�>>ก]GQE�f� 2 ก��K�GQ��ก���Y>AQ>�J�กK>��������ก���G ACTH (Before ACTH injection) ��B�I�� �����ก��G�> ��G�Q� (After ACTH injection) W�� C��KRG�E������กก��@�>GDEC�E���RG�����]�กK>��������ก���G ACTH ����G��o>�?R���>�?��W>�RG�E�����E�Kก�� 14.30± 4.14 ng /g of dry feces (N=4) ��I�� �ก�����ก��G�> ��G ACTH �Q�C��K��c��DTIQ��G��A> �>�?��W>�EC���A�S�>�ก�����ก�K 20 ng/g T�NK� 3 ����ก �K>�ก��S���G�����EAQ�AQ�A> o>�?R�����G� ก�K��G���ก�� ��EAQ@JKB�����E�f��ก��B�T� 1 @��GI? (@G G� BC��� 4) ��E�Y�>�DAQ>�J���������EAQ�AQ�A> o>�?R���>�?��W>�T��กก��@�>GD���]�K]GQ����JK�E�����E����ก��NK� �����ก���G ACTH C��K �กก��@�>GD���]�K]GQ����JK�������o>�?R��E�����Tก�QE��� ก���กก��@�>GDEC�E�������]�กK>��������ก���G ACTH (�ก�K >�K ]�K�����@D��U� @_��� P>0.05) �KE�Y�>E�����E����ก��B�I�� �����ก���G ACTH C��K���������o>�?R����Dก�K>�K �����@D��U� @_��� P<0.05 ) G� �� ��� 2

����,��� 2 @G ��G�����EAQ�AQ�A> �>�?��W>�E�����T����>�K �J�A> �กก��@�>GDEC�E��� (Means for Cortisol metabolites quantity of the females evaluated)

N��G@���?

(Name of individuals) @_�BC (Status)

�KE���������� Cortisol metabolites (ng/g of feces) �D��� (N) mean±SD

ก��@�>GD EC�E��� ]�K]GQ����JK 28 15.15 ± 6.44 a ก��@�>GD EC�E��� Before ACTH injection 4 14.30 ± 4.14 a ก��@�>GD EC�E��� After ACTH injection 7 28.32 ± 13.60 b

I��EI�� a �� b @G ���EI�Y>�I�Y>����ก�K ��I�K ก��K�>�K �����@D��U� @_��� E�Y�>E�����E��������ก�K �KE�������I�K �JKA> I�K��ก��G�> RG���P� LSD �� Duncan�s �����G�����ENY�>���� 95 %

��)��� 4 ก�p@G ���E��Y�>�]I�A> ��G��o>�?R���>�?��W>� (Cortisol metabolites)

T�NK� �����ก���G ACTH

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4. ��7#_2ก���'-�5 RG�����Q��กก�������G��G�����EAQ�AQ�A> o>�?R��R��E�@E�>R��T��กก��@�>GDEC�E������]�K����JK���

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@K��c�ก���E���I?@B��ก��D �A> �K>�]�Q�K>I�Y> Adrenal GQ��ก�������Go>�?R���>�?��W>�C��K ����G�����EAQ�AQ�A> o>�?R���>�?��W>�E����� (Average baseline) E�Kก�� 15.15±6.44 ng/g of dry feces (C�@�� 3.56-26.34 ng/g) W��

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E�Y�>�G@>����@J ��DA> ��G�����EAQ�AQ�GQ��ก�E�����E����ก��ก�ก����Q�TIQ��ก�@� E���I?o>�?R���>�?��W>�RG��G�> ��G ACTH C��K �K>�]�Q�K>�� � @G _� B������� � �D �>�JK (active) ��S ��S��G�����EAQ�AQ�A> �>�?��W>�T��กก��@�>GDEC�E�������� ]�K����JKT��r�������� � >�JKT���G�����]�K@J c�G�ก���K>�K TG

5. 6/0%��01�$ �กก���ก�T�ก�������กก��@�>GDEC�E������]�K����JKG� ก�K���S�� � @G _� @B�������ก��D �A> �K>�

o>�?R��EC� (active) >�JK RG���ก�E��Y�>�]I�A> o>�?R��R��E�@E�>R�� ���������o>�?R���>�?��W>�>�JKT���G���ก�� �K�r������ (EGY>�ก����� 2553) W�� Tก�QXGJc@�C��P�?����D�� �กก��@�>GDEC�E���G� ก�K��� � ����R�Q������]�K����JK GQ��EI����S�� �DE�f�������Q> ��ก��r����>Y��V ���Eก����AQ> �K>ก�����JK�K>]� RG�E�C�T�E�Y�> A> ก���ก�CX��ก�����ก�GDE���ก����E�f�ก�@K E@���CX��ก���ECY�>ก�����JK��S T�EC�cJQ��EC�E��� G� ENK� ก�GDE���ก���ก�CX��ก�����E�����JK�ก��ก������E�����@����@DE�e�E�Y�>I����กK>� >��ก���Gก�ก� E��S� ENK� ก���G�DI�K ก� EC�E��S� I�Y>ก��> ����E������CYS����ก� E��S� ECY�>ก�E�����JKTIQ�����EI��@��กA�S� TIQE�f�ก�@K E@���CX��ก������@��C��P?ก��K�RG�E�C�CX��ก������Eก����ก���� TIQ���กA�S� E�f�>�กI�� T�E�YS> �Q����@��_�D]GQ K����@�G �>ก�ก��Sก���Gก����@���?GQ��ก���GII�Y>�กE������@�C��P�?กe�� � E�f�@�� @D��U������Q> ��ก�GDE���ก�T�>���>�JK�K>]�.

6. %0ก���0/�,0', Brown, J., Walker, S. and Steinman, K. 2004. Endocrine manual for the reproductive assessment of domestic and non-

domestic species, 2nd edition. USA : Smithsonian institution. Rupert Palme. 2005. Measuring Fecal Steroids Guidelines for Practical. Ann. N.Y. Acad. Sci. 1046: 75 80

Rupert Palme. 2005. Measuring Fecal Steroids Guidelines for Practical Application. Academy of science, New York. Scott E. Cortisol and stress: How to stay healthy. [online]. Available from: http://stress.about.com/od/stress

health/a/cortisol.html. : 2006 Toni E. Ziegler, and Daniel J. Wittwer. 2005. Fecal Steroid Research in the Field and Laboratory Improved Methods for

Storage, Transport, Processing, and Analysis. University of Wisconsin. American Journal of Primatology, 67 : 159-174.

Tusanee Apichartsrungkoon. 2002. Effect of stress on Progesterone and Luteinizing Hormone in Female Reproductive System. Journal of Agriculture , 18 (3) : 261 269.

Unknown. Cortisol . [online]. Available from: http://en.wikipedia.org/wiki/Cortisol : 2010

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Use of fecal hormones assays for evaluating progesterone patterns in Females

Rucervus eldi siamensis and Rucervus eldi thamin. .

�5P�,(� #QR�(,1, �7�*'2�กUP� &����$�&1, �'�5� %) ��7ก�1, 0�&)'ก� �0,��ก.�2, �'�*���� �0,�')5�['�'%. 3 12$ 3�')��� ['�'0�7P�����2 Chainarong Punkong, Suttiluck Meeveerasom, Nittaya Petsukorn, Ampika Thongphakdee, Sittawee Thongtipsiridech

and Boripat Siriaroonrat

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(Progesterone) T�ก��K������ C��P�?]��������� C��P�?C�KEC�E��� C�ก�E�������� A> o>�?R��R��E�@E�>R�����@G _� � �>�ก��D �A> �� ]AK (ovarian cyclicity) RG�T������ C��P�?]�� ������� �>�ก�E�f�@�G (estrous cycle) ��E����� 21.64±1.83 ��� (±SEM, C�@�� 12-32 ���; n=11 cycles) ���K��������EAQ�AQ�CYS�u� (baseline concentration) A> o>�?R��R��E�@E�>R�� E�����E�Kก�� 298.91±11.99 ng/g of dry feces ��S ��S�K�����o>�?R�����@G _� ���� luteal phase ���K>�JK��I�K 448 _� ก�K 3,000 ng/g @J @�G��� 3,112 ng/g of dry feces ��NK� ����E�����E�f� follicular phase �� 5-9 ��� ������E�f�@�G�� 1-2 ���

T������ C��P�?C�K C��K��� �>�ก�E�f�@�G��E����� 20.75±1.13 ��� (±SEM, C�@�� 14-30 ���; n=20 cycles) �K��������EAQ�AQ�CYS�u�T�������]�K��S �Q> E�����E�Kก�� 172.0±28.09 ng/g of dry feces �����EAQ�AQ�A> o>�?R��T����� luteal phase ���K>�JK��I�K 258.96 _� 1,840 ng/g of dry feces ���กก���ก������ C��P�?C�K��ก���S �Q> �D��� 3 ��� T��D�����SE�f������ �����S �Q> @��J��?]GQ�Jก���ก�� 1 �������S �Q> ]�K@��J��? 2 ��� ������E����S �Q> �ก���� 34 @��GI? RG�T�NK� @��GI?�กV ���������o>�?R��R��E�@E�>R��>�JK�����G������� 400-1,000 ng/g ��@J A�S�E�Y�>�V >�K EIe�]GQN�G (RG�E�C�T�NK� 23 @��GI?@�G�Q�กK>���>G) ��S ��SC��KT�NK� @��GI?�Q�V �����o>�?R�����C�T������ C��P�?C�K�����S �Q> ��@J ]GQ_� 14,720 ng/g of dry feces �����G��D� T�NK� @�G�Q�A> ก���S �Q> W�� �กก���ก�ก�E�������� A> �����o>�?R��R��E�@E�>R��T�NK� XGJก�@Y�C��P�?G� ก�K� ��E�t���@ �?���@D��U�K>ก��D]�TNQE�f�u�AQ>�J�>Q >� @DI���ก�� c����I���Gก�ก�EC�E��S� A��C��P�? (breeding management plan) GQ��ก������ก�?TNQE������K V]GQ>�K EI��@��ก��� A�S�T�R>ก@�K>V ]� (V��V�(�:: ����� C��P�?]��; ����� C��P�?C�K; �K���EAQ�AQ�CYS�u�o>�?R��R��E�@E�>R��; ����ก���S �Q> A> �����

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ABSTRACT The fecal hormones assays for evaluating progesterone patterns in females Rucervus eldi siamensis and Rucervus eldi thamin were found that all of studying females have an evidence of ovarian cyclicity. The average estrous cycle length for all Rucervus eldi siamensis was 21.64±1.83 days (±SEM, range 12-32 days; n=11 cycles). Overall baseline concentrations of fecal progesterone averaged 298.91±11.99 ng/g. In the luteal phase, concentrations ranging from approximately 448 to 3,000 ng/g with peak concentrations reaching as high as 3,112 ng/g. The follicular phases (i.e., when progesterone concentrations were at baseline) generally lasted 5-9 days, behavioral estrus were approximately 24-48 h duration.

The average estrous cycle length for all Rucervus eldi thamin was 20.75±1.13 days (±SEM, range 14-30 days; n=20 cycles). Overall baseline concentrations of fecal progesterone averaged 172.0±28.09 ng/g. In the luteal phase,concentrations ranging from approximately 258.96 to 1,840 ng/g. One complete and two partial pregnancies were monitored during the study. This Gestation duration was 34 wk with fecal progesterone concentrations remained at 400-1,000 ng/g for the first several weeks, then increased markedly during pregnancy (especially the last 23 wk) with peak concentrations reaching as high as 14,720 ng/g before an abrupt decline coincided with parturition. Finally, from study of fecal progesterone variation in breeding season by determination reproductive status (ovarian activity, estrous cycle, baseline concentrations and pregnancy diagnosis) has contained an important purpose for data base requirement to breeding management plan by assisted reproductive technologies to be appropriate. Keywords: Rucervus eldi siamensis; Rucervus eldi thamin; progesterone baseline concentrations; gestation duration of Eld�s deer

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E���ก�K �non-invasive hormone monitoring� RG�>���ก�Eกe����>�K >���� (fecal samples) ��Dก���E���I?I�����o>�?R�� (Plame, 2005)

T�ก�������G�K@���?EC�E�����S���ก���S �Q> E�@��_�Dก��Y����c��������กDI�G����E��ก���>G]GQ�กก�������E���I?�����o>�?R�� W�� ก�������������ก���S �Q> GQ��ก��D>����QW��? (ultrasound) ��S�_Y>�KE�f���P����������K��D�ก���@�G �K>�K ]�กe��]�KTNK@���?��กN��GI�Y>��ก������@��_�����Dก�C�@J��?���GQ����P�G� ก�K�]GQ G� ��S��� Eก�Gก������ก�?TNQก�������G�����o>�?R����S T��r@@����>��������@��_�D��Y����c�ก���S �Q> ]GQ RG�NK� E��A> ก���>GI�Y>�ก�Jก>�@��_�Dก������]GQ_Q�������E��ก���S �Q> ����K�>�A> @���?N��G��S�V ����ก�@� Eก�C�NK� A> ก�E�����Q�c@�C��P�?�K��ก��ก��AQ>�J�ก�������G�������o>�?R�������>�����_� T�NK� ����E��G� ก�K� ��S ��ST�ก�� c�TIQ��ก���G@G �K��ก����I�K EC�E���ก��EC�cJQ��S� @��_ก���DTIQ]GQ���R�N�?@J @�GGQ��ก�� c�ก�Eกe����>�K >��������G� ECY�>C�@J��?����KCYS�u������o>�?R�� (baseline hormone) A> EC�E����K��N��GI�Y>�K����� W�� ก�Eกe����>�K I�Y>GDE���ก���ก�>�K �K>E�Y�> ���DTIQ����T�]GQ�Kก��D �A> �� ]AK (ovarian activity) T�NK� ��S�V �����C�Q>��K>ก�c@�C��P�?GQ��ก�TNQก�����ก���Gก�ก� E��S� ����JKTIQ>�JKก��EC�cJQ]GQ>�K _JกNK� E�� (Morrow, 2007) I�Y>A��C��P�?@���?GQ��ก�TNQE������K V >��ก�c@�E����GQ���SDENYS> ก��Q�<ก���>K>�T��K��� ���_� E�����>Y��V E�f��Q�.

2. ���W7#�$�,(� 2.1 ECY�>������G��ก�E�������� A> ��G��o>�?R��R��E�@E�>R��T������ C��P�?]��������� C��P�?C�K RG�

��P�ก�@ก�G�ก>����� 2.2 ECY�>���E���@BC� @�������A> ����@Y�C��P�?T������ C��P�?]��������� C��P�?C�K 2.3 ECY�>TNQE�f�u�AQ>�J�>Q >� T�ก���Gก�ก� E��S� ��C�@J��?ก�E���UC��P�?T�ก��K����Nก������ �K>]�

3. �'*�ก��[\กU� 3.1 ���05��,�����12$ก��%กi3���05��,&92����� (Animals and Fecal Sample Collection)

����D���@���?���TNQT�ก���ก���S ���D�����S @�S� 10 ��� E�f�EC�E�����S I�G �ก@��@���?��I�K�� �B���u��� 3 IK (���� ��� 1) RG�E�f������ C��P�?C�K �D��� 8 ��� ������� C��P�?]���D��� 2 ��� W�� _JกTNQECY�>ก�C�{�E��R�R���� ก�@Y�C��P�?��ก�c@�E���� I�Y> artificial insemination (AI) ��ก��Q�<ก���>K>� I�Y> embryo transfer (ET)

�Dก�Eกe����>�K �J�A> ����� �K����� (individual fecal sample) 10-50 g @��GI?�� 1-3 ���S E�f�����E�� 6-10 EGY>� (��I�K NK� EGY>� E���� 2553-�ก��� 2554) W�� I�� �กก�Eกe����>�K ]GQ���D]�Eกe���ก�@BC���>��IBJ��-20 > �EW�EW��@ ��ก�K���D]�EAQ@JKก�����ก���E���I?

3.2 ก���ก�.%���5�05.�j0���&�-�ก&92����� (Fecal Steroid Extraction)

�D���>�K �J�@���?���NKAe ]�Q��DTIQIQ GQ��ก��DEAQ�JQ>�����Q>� (hot air oven) ���>��IBJ�� 60 > �EW�EW��@��IQ ���TIQ�กE�f�c Eกe�EAQ�JQNKAe ��ก�@BC��� -20 > �EW�EW��@ ��ก�K���D]��Dก�@ก�G �Dก�@ก�GRG�N�� �SDI��ก�J�@���?���IQ E�f�c 0.1 ก��� T@K� T�I�>G�G�> (test tube) E��� 90 % E>���>�ก>o>�? 5 ��������� (ml.) �Q��D]��Q�E�f�E���� 20 ��� I�� �ก��S��D]��r��EI���� ������E�e��>� 2,500 g E�f�E���� 20 ��� ���Eกe�E�C�@�����T@V GQ��� �ก��S��D��ก>����EI�Y>]��D����TI�K>�ก���S T�E>��>� 90 % 5 ��������� �D@��������E�f� extractans

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ก�� ���ก��� ������ ����� 2 ������� 1 ����� ก ����� 2554

]� vortex �� 30 ����� �Q��D]��r��EI���� ��� 2,500 g E�f�E���� 15 ��� ���@�����T@V GQ������E�f� supernatants ���ก��ก���>��ก�D]��D dry �Q��D����>�ก�>� (redissolv) RG�ก�����T�E>��>� I�Y> dilution buffer (>�K TG>�K I��� ) 1 ��������� �D extractans ]� vertex �� 1 ��� Eกe�NKAe ���>��IBJ�� -20 > �EW�EW��@ ��ก�K���D]��Dก�������E���I? (assay) W�� ก�����ก�@ก�GGQ����P�ก��Q� ��Tก�QE��� ก����S�����@��P�BCT�ก�@ก�GE>E@����>�G?o>�?R��]GQ�กก�K 85 % A�S�]� (Extraction recovery efficiency was >85% for all steroids.) (Brown et al., 2004)

3.3 ก�����-�'%(��$+�#�'&�Pj0���&�

�Dก���E���I?GQ����P� Enzyme immunoassay �� Competitive ELISA (Enzyme Linked Immuno Sorbent Assay) RG�TNQI��กก��K ก�����ก�� Monoclonal Antibody: Pregnane CL-425 (Provided by Quidel Corporation) ��I�K >���E�� (antigen) I�Y>o>�?R��R��E�@E�>R�� E����>]�E�@ (progesterone metabolites)T����>�K ��S�V ก�� enzyme conjugate: Pregnane Horseradish Peroxidase (HRP) �Q��DTIQEก�G@�GQ��ก�TNQ@����� substrate (ABTS and catalyst: Hydrogen peroxide) �D���ก����ก�� HRP-labeled hormone ��G�K���EAQ�@ ���Eก�GA�S����D�����������EAQ�AQ�GQ��E��Y�> @E��R��RpR���E�>�? (spectrophotometer) ����������Y�� 405 �R�E��� (nm.) (Brown et al., 2004)

����,��� 1 @G ��NY�>I�K�� �I�Y>@_���EC�E��S� ����� ����D�TNQT�ก���ก���S (Summary of institutions with Eld�s Deer participating in this study)

Institutions 2$&��,)��*7�T�5

(Rucervus eldi siamensis)

2$&��,)��*7�)&�� (Rucervus eldi thamin)

N (M/F) Age (yr)* N (M/F) Age (yr)*

@��@���?E�FGEAEA��� - - 0/3 -/-/-

@��@���?G�@�� 0/2 -/- - -

�I�������Eก����@��? (กDC @� **)

- - 0/5 -/-/-

*: AQ>�J�_� �� 2553 ** ]GQ�������� �ก@��@���?E�FGEAEA���

3.4 ก���'%(��$+�6/0&92 (Data analysis) �KE�����A> AQ>�J�T������S�DE@�>T��J��� �KE�����±�����GE��Y�>����u� (Mean±SEM) W�� ����A> �D�K

�Estrous cycle� >�JK��CYS�u�A> ��c�I�Y>R�� �K (profiles) A> o>�?R��R��E�@E�>R���ก>����� (fecal progesterone) ��S ��ST�ก���A> @���?���]�K]GQ��S �Q> (non pregnant) �K��G�����EAQ�AQ�CYS�u� (baseline) o>�?R��R��E�@E�>R�����������G]GQ @��_�D���]GQ�กก��D�KE��������]GQ�กก���E���I?���>�K @> ���S �I�KE�����A> �J��K�����>�K �Q��D�KE�����A> �K�����>�K ���]GQ�I�KE�����WSD>�ก (recalculated) ��GE>�K���Eก���ก�KE�������กGQ�� 1.5 E�KA> �KE���� E�����u� (1.5 SD) >>ก]� ���Dก��D���TI�KWSDV ����S>�กI�����S ��ก����� ]�K���KTG���@J Eก��ก�K�KE�������กGQ�� 1.5 E�KA> �K SD (Brown et al., 1994)

T�ก�กDI�G�������E�f� luteal phase ��S� ��E�����ก��GE�����Q������G�����EAQ�AQ�A> o>�?R��@J A�S��ก��G�� baseline ����� 50 E�>�?EWe��?��� ��G�����@J �K>E�Y�> RG���G@�S�@�GA> luteal phase ������ก��G����K��G�����EAQ�AQ��G� �>�JKT���G�����EAQ�AQ�CYS�u� (baseline) >�ก���S @K��� �>�ก�E�f�@�G (estrous cycle) �����������E�K]I�K��S�

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ก�� ���ก��� ������ ����� 2 ������� 1 ����� ก ����� 2554

@��_�D���]GQ�ก��GE����A> ก�E�f� luteal phase >��I��� ]�_� ��GE�����Q�>�ก>��I��� ����K>E�Y�> ก�� (Brown et al., 1994) @K��B�����E�f� anestrus I�Y>B������ �>�� EC���ก�I��G� ��� N������ (sexual cycle rest) ��กDI�GTIQ>�JK��I�K ����A> ก�E�f� luteal phase ���C�ก�������ก�K 2 E�KA> NK� E���ก��A> �������E�f� follicular phase

��S ��ST�ก���ก���S��ก�K�_� ก�E�������� ���E�f�� �>��� estrous cycle I�Y> cyclic changes A> o>�?R��

R��E�@E�>R�� (progesterone) RG�� �>�ก�E�f�@�GT������ ����]���>�JK�������� 21 ��� I�Y>>�����]GQ_� 30 ��� @K��

����E��ก���S �Q> T������ E����� 33.5 @��GI? (Monfort et al., 1990)

4. _2ก��[\กU� �กก�������E���I?o>�?R��T�>�����ECY�>���E�����c����E�f�� �>� ��ก�E�������� A> o>�?R�� R��E�@E�>R��T�ก��K������ C��P�?]��������� C��P�?C�KEC�E��� (������_� ก��K�������Q�<ก���>K>���c@�E����GQ���SDENYS>NKAe ) GQ����P�ก�@ก�G�� boiling method ���Dก�������G�����o>�?R��GQ����P� Enzyme immunoassay @��_�D�กc�ก���ก�]GQG� �K>]���S 4.1 2$&��,)��*7�T�5 (Rucervus eldi siamensis) �กก���ก�ก�E�������� A> ��G��o>�?R��R��E�@E�>R��T����>�K >����� (fecal progesterone) A> ����� C��P�?]��EC�E����D��� 2 ��� C��K��G��o>�?R��R��E�@E�>R��@��_@G _� ก�E�������� A> �� ]AK���E�f�� �>�]GQ>�K N�GE�� (ovarian cyclicity) RG���NK� �����������A> � �>�ก�E�f�@�G (estrous cycle) E����� 21.64±1.83 ��� (±SEM, n = 11 cycles) RG�NK� ����A> ก�E�������� ���E�f�� �>��������������>��KNQ>>ก]� W�� >�JKT�NK� (C�@��) 12-32 ��� �K��������EAQ�AQ�CYS�u� (baseline concentrations) A> fecal progesterone T������ C��P�?]��EC�E������KE����� (Mean±SEM) E�Kก�� 298.91±11.99 ng/g RG�����������EAQ�AQ�A> o>�?R�����@G _� �������E�f� luteal phase >�JK��I�K 448 _� �กก�K 3,000 ng/g ��S ��S��G�����EAQ�AQ�@J @�G (peak concentration) @J _� 3,112 ng/g of dry feces (BC��� 1. a, b) W�� NK� �������E�f� follicular phase (E�Y�>����� progesterone � �>�JKT���G��A> baseline) ������� 5-9 ��� �>ก�ก��SCX��ก������@G _� B��A> ก�E�f�@�G (behavioral estrus) ������E������ 1-2 ��� RG���>GNK� ���������ก���ก������o>�?R�� (CX�B��-ก����� 2553) ]�KC��K����� C��P�?]����S @> ���@G _� B�����E�f� anestrus �K>�K TG ��]�KC�ก���S �Q> A> ����� C��P�?]����S @> ���

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��)��� 1. Individual profiles of fecal progesterone concentrations in two representative Eld�s Deer females (Rucervus eldi siamensis). Reproductive cycles are designated by an asterisk. (single asterisk represent normal cycle and double asterisk a long cycle ; * normal, **long cycles)

4.2 2$&��,)��*7�)&�� (Rucervus eldi thamin) �กก���ก�ก�E�������� A> ��G��o>�?R��R��E�@E�>R��T����>�K >����� (fecal progesterone) A> ����� C��P�?C�KEC�E���C��K��ก�E�������� A> �� ]AKE�f�� �>� (ovarian cyclicity) ENK�ก�� RG���NK� �����������A> ก�E�f�@�G (estrous cycle) E����� 20.75±1.13 ��� (±SEM ,C�@�� 14-30 ���; n= 20 cycles) @DI����K��������EAQ�AQ�CYS�u� (baseline concentrations) A> fecal progesterone ���KE����� (Mean±SEM) E�Kก�� 172±28.09 ng/g RG�����������EAQ�AQ�A> o>�?R��R��E�@E�>R�� (progesterone) ���@G _� ���� luteal phase >�JK��I�K 258.96 _� ก�K 1,800 ng/g of dry feces ��S ��S��G�����EAQ�AQ�@J @�G (peak concentration) @J _� 1,840 ng/g of dry feces (BC��� 2. a, b and c) RG���>GNK� ���������ก���ก������o>�?R��G� ก�K�C��K����� C��P�?C�K 3 ����ก 8 ���@G _� ก���S �Q> RG�E�f�ก���S �Q> �กก�c@�E����GQ���SD ENYS>NKAe ��ก�������� �A> ���@���?C��?�I�������Eก����@��? ����EA�กDC @� 1 ��� >�ก 2 ��� E�f������ ���c@�E����T�I�>GกQ��Q��Q�<ก���>K>���ก�������� �A> ���E�QI�Q���> �?ก�@��@���?v �DRG�@D��ก>����ก�? ����� ��ก���ก� ��S ��S@��_�D�กc�ก���ก������o>�?R�� progesterone ]GQG� �K>]���S

4.2.1 2$&��,)��*7�)&�����_�&%��5&./�5ก��8 /�4V�% ̀401 �16i, �กก�������G�����o>�?R�� progesterone T�NK� ����������� C��P�?C�KG� ก�K�]GQ���ก�c@�E����RG�ก�

@>G�SDENYS>NKAe ���������o>�?R�� progesterone >�JKT���G����DV (nadir levels) RG��G�K��E�f�NK� ���@���?>�JKT�����A> ก�E�f�@�GI�Y>��]AK�กC�Q>��K>ก�_Jกc@�GQ��ก���G�SDENYS> (�������o>�?R�� progesterone ≤ 300 ng/g of dry feces) W�� T���I�K NK� �����ก���S �Q> ��S�C��K�����o>�?R�� progesterone ��� >�JKT���G��@J ก�K��Y>T�NK� ����E�����Q�A> ก���S �Q> ��G��o>�?R����� >�JKT���G������� 400-1,000 ng/g (NK� 1-11 @��GI?�ก) ��@J A�S�E�Y�>�V >�K EIe�]GQN�G (> 1,500 ng A�S�]�) RG�E�C�T�NK� 23 @��GI?@�G�Q�กK>���>G ��S ��SC��KT�NK� @��GI?�Q�V �����o>�?R�� Progesterone ���C�T������ C��P�?C�K�����S �Q> GQ��ก�c@�E������@J ]GQ_� 14,720 ng/g of dry feces �����G� ��_� ��G����D@�GT�NK� @��GI?@�G�Q�A> ก���S �Q> W�� RG�@����Q�C��K����� C��P�?C�K���]GQ���ก�c@�E����GQ���SDENYS>NKAe G� ก�K�������ก���S �Q> �� ����� 34 @��GI? (BC��� 3.)

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��)��� 2. Individual profiles of fecal progesterone concentrations in three representative Eld�s Deer females (Rucervus eldi thamin).

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��)��� 3. Fecal progesterone concentrations in an individual Eld�s Deer female (Rucervus eldi thamin) throughout gestation.

4.2.2 2$&��,)��*7�)&������V�ก��5/�5��ก���0�0� �กก�������G�����o>�?R��R��E�@E�>R�� (progesterone) T������ C��P�?C�K���]GQ���ก��Q�<ก���>K>�

B�I�� ก��Q�<ก (�Dก��Q�<กNK� ������ 30 ����� 2553) C��K����� C��P�?C�K��S @> ��������S �Q> ���������o>�?R��>�JKT�NK� 200-5,000 ng/g of dry feces RG�T�NK� ����� 11-15 @��GI?�กA> ก���S �Q> ���������o>�?R�� progesterone E�����>�JKT�NK� 200-1,000 ng/g of dry feces I�� �ก��S������o>�?R�� progesterone ��EC���@J A�S� E������กก�K 1,000 ng/g of dry feces ��T�NK� @��GI?��� 21-23 �����o>�?R�� progesterone ��EC���_� ��G������� 3,000-5,000 ng/g of dry feces ���G��G�����EAQ�AQ�� T�NK� 1-2 @��GI?@�G�Q�กK>���>G ��S ��SC��K����� �Q�<ก���>K>�G� ก�K�������E��ก���S �Q> ��E����� 31 @��GI? RG��Jก����� Eก�GTI�K��A����>G��S @> ��� (BC��� 4. a and b)

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��)��� 4. Fecal progesterone concentrations in two Eld�s Deer females (Rucervus eldi thamin) throughout gestation

from ET techniques.

5. ��7#12$6/0%��01�$ 5.1 ��7#0�'#��5_2 �กc�ก���ก�ก�������E���I?o>�?R��R��E�@E�>R��T�>����� (fecal progesterone) ECY�>���E�����c�ก�E�������� A> o>�?R��R��E�@E�>R�� (progesterone) T�ก��K������ C��P�?]��������� C��P�?C�KEC�E��� C��KT�T������ C��P�?]��������� C��P�?C�K��ก�E�������� A> �� ]AK���E�f�� �>�RG���NK� �����������A> ก�E�f�@�G (estrous cycle) E����� (Mean±SEM) E�Kก�� 21.64±1.83 ��� (C�@�� = 12-32 ���) �� 20.75±1.13 ��� (C�@�� 14-30 ���) ���DG�� W�� ���KTก�QE��� ก�� @DI����K��������EAQ�AQ�CYS�u� (baseline concentrations) A> fecal progesterone T������ ��S� C��KT������ C��P�?]����C��P�?C�KEC�E�����������ก�K ก��>�K �����@D��U� @_��� (P<0.05) ก�K��Y>T������ C��P�?]�������KE����� (Mean±SEM) A> fecal progesterone ���E�f� baseline E�Kก�� 298.91±11.99 ng/g W�� �กก�K���C�T������ C��P�?C�K������KE����� E�Kก�� 172.0±28.09 ng/g ��S ��SC��KT������ C��P�?C�K�����ก���S �Q> ��]GQ�Jก�������ก����ก�� (�กก�c@�E����GQ���SDENYS>) ��������ก���S �Q> ������� 34 @��GI? RG���>G����A> ก���S �Q> �������o>�?R�� progesterone >�JKT�NK� 400 _� ����� 14,000 ng/g of dry feces W�� T�NK� ����E�����Q�A> ก���S �Q> ��G��o>�?R����� >�JKT���G����D ����@J A�S�E�Y�>�V RG�E�C�T�NK� 23 @��GI?@�G�Q�กK>���>G �����G� ��_� ��G����D@�GT�NK� @��GI?@�G�Q�A> ก���S �Q> T�ก���A> ����� �����S �Q> GQ����P�ก��Q�<ก���>K>�T�ก���ก�G� ก�K���SC��K��������S �Q> ���@�S�ก�K����� ���]GQ���ก�c@�E����GQ���SDENYS>NKAe ก�K��Y>������ก���S �Q> ��E�����EC�� 31 @��GI?��E�Y�>�Dก�E�����E������G�����EAQ�AQ�A> �����o>�?R�� progesterone C��K������ก�K ก��>�K �����@D��U (P<0.05) RG������ �����ก��Q�<ก���>K>�T�ก���ก�G� ก�K�����G��A> o>�?R��ก���S �Q> ����K>�AQ ��D EC�� 200-5,000 ng/g of dry feces W�� ��GE�f������ ½ A> ����� �����ก��Q> �Q> GQ��ก�c@�E���� ��S ��SC��K�Jก����� Eก�GTI�KGQ����P�ก�G� ก�K�����ก�������� ]�KE�f�]����ก������A����>G@>G��Q> ก���J���A> ก�E�������� �����o>�?R�����E�f�]�T��J�������K>�AQ �����������@J W�� @EI��A> ก�E�������� �����o>�?R�����c�G�ก��G� ก�K�����R�Q������Eก����AQ> @��C��P?ก��ก�EC���A�S�A> �����o>�?R�����E����G

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(cortisol) ���E�YS> �Q�C��K��ก�EC���A�S�c�G�ก����I�K NK� �����ก���S �Q> T������ �Q�<ก���>K>���S 2 ��� ��S ��S@EI��A> ก��������o>�?R�����E����GT���G��@J >���I���r�������Eก����AQ> ������Q> �Dก�กQ]A������� T�ก���ก��K>V ]� �>ก�ก��SE�Y�>�Dก�C����E�����E����c�ก���ก���G��A> o>�?R�� progesterone T�ก���ก�G� ก�K���SE����ก��ก���ก���S T����K ���E�� C��Kก���ก�ก�E�������� A> o>�?R�� progesterone T������ C��P�?]�� (Rucervus eldi siamensis) G� �J���G� ก�K�E�f��� �ก���ก����S �ก W�� �� �ก���ก�@K��TIUK���C���E�f�ก���ก�T�ก��K������ C��P�?C�K (Rucervus eldi thamin) ��S ��SE�Y�>�Dก�C�����������A> ก�E�������� o>�?R�����E�f� estrous cycle A> ก��K������ C��P�?]����C��P�?C�KC��KRG�E��������������������� 21 ��� �>ก�ก��S�กก���ก��� C��K� �>�>�����]GQ_� 30-32 ��� @>G��Q> ก���� �A> Monfort et al. (1990) �������_� � �>����E�f� estrous T������ ����]���>�JK�������� 21 ��� ������]GQ_� 30 ��� ��E�Y�>�Dก�C�����J���ก�E�������� A> �����o>�?R�� progesterone T������ �����ก���S �Q> ��]GQ�Jก�������ก����ก����S�C��K����ก���S �Q> (34 @��GI?) ��NK� @��GI?�����ก�EC���A�S�A> �����o>�?R�����EIe�]GQN�G (��S �K@��GI?��� 23 A�S�]�) ���KTก�QE��� ก�� RG�@����Q���S ����� C��P�?]��������� C��P�?C�K��S���GE�f�@���?�����NK� XGJก�@Y�C��P�?������� ��S ��ST�I��� XGJก�@Y�C��P�? (breeding or reproductive season) @��_��� �>�ก�E�������� A> �� ]AK]GQI�����S (polyestrous) ��S ��S����R�Q��K����� C��P�?C�K� ���T�ก���ก�G� ก�K� @G _� NK� C�กI�Y>NK� ���E�f� anestrous T���I�K NK� EGY>������_� �ก��� (BC��� 2. b and c) ��EGY>��ก���_� ����� (BC��� 3) Tก�QE��� ก���� �ก���ก�A> Monfort et al. (1990) ��������KXGJก������ก�E�������� A> �� ]AK��E����T�NK� EGY>��ก���_� ����� ��@�S�@�GT�NK� EGY>�@� I��_� ����� �>ก�ก��S Hosack et al. (1997) ]GQ�� ��K����� C��P�?C�KEC�E���T�ก� E��S� A> >E���กEI�Y> ����XGJก�@Y�C��P�? (reproductive season) ��E����� 277.9±9.7 ��� ����� �>�ก�E�������� A> �� ]AKE����� 13.8±0.5 � �>��K>XGJก� RG�>���GE�f��������A> � �>������ 19-21 ��� W�� Tก�QE��� ก��ก��ก���ก����]GQก�K�_� AQ �Q���ก���ก�T����S ��S

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�����ก���D���D]�TNQ>Q >� I�Y>�D]�TNQ���R�N�?T�ก���Gก�ก� E��S� TIQ�����@��P�BC]GQW�� ����������ก�@���@���ก�GDE��� �G� ก�K�>�K �K>E�Y�> �>ก�ก��ST�ก���ก�G� ก�K��� ]�K��>�������>GNK� XGJก�I�Y>�������A> XGJก�E�Y�> �กAQ>�Dก�GA> �D������>�K �J�@���?���]GQ���������E��T�ก�GDE��� � W�� >�@��_�D�C����T�ก�� c�ก���ก�T�R>ก@�K>V ]�.

6. %0ก���0/�,0', Brown, J.L., Wasser, S.K., Wildt, D.E. and Graham, L.H. 1994. Comparative aspects of steroid hormone metabolism and

ovarian activity in felids, measured non-invasively in feces. Biol Reprod. (51): 776-786. Brown, J.L., Bellem, A.C., Michael, F., Wildt, D.E. and Roth, T.L. 2001. Comparative Analysis of Gonadal and Adrenal

Activity in The Black And White Rhinoceros in North America by Noninvasive Endocrine Monitoring. Zoo Biology. (20): 483-486.

Brown, J.L., Walker, S. and Steinman, K. 2004. Endocrine manual for the reproductive assessment of domestic and non-domestic species, 2nd edition, Smithsonian institution. USA.

Hosack, D.A., Miller, K.V., Marchinton, R.L. and Monfort, S.L. 1997. Ovarian Activity in Captive Eld�s Deer (Rucervus eldi thamin). Journal of Mammalogy. 78(2): 669-674.

Monfort. S.L., Wemmer, C.M., Brown, J.L. and Wildt, D.E. 1990. Use of urinary hormone assays for evaluating endocrine patterns associated with the long-day breeding season in Eld�s deer (Rucervus eldi thamin). J. Exp. Zoo Suppl., (4) : 215-8.

Morrow, C. 2007. FAQ - Frequently Asked Questions. [online]. Available from: http://scimitarscientific.com. [2011, March 15].

Palme, R. 2005. Measuring Fecal Steroids Guidelines for Practical Application. Academy of science, New York. Schwarzenberger, F., Walzer, C., Tomasova, K., Vahala, J., Meister, J., Goodrowe, K.L., Zima, J., Strauß, G. and Lynch, M.

1998. Fecal progesterone metabolite analysis for non-invasive monitoring of reproductive function in the white rhinoceros (Ceratotherium simum). Anim Reprod Sci, (53): 173-90.

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Progesterone patterns in Siamensis Eld�s deers (Rucervus eldii siamensis) and Thamin

(Rucervus eldii thamin) assessed by fecal hormone assay

Chainarong Punkong1, Suttiluck Meeveerasom1, Nittaya Petsukorn1, Ampika Thongphakdee2, Sittawee Thongtipsiridech3 and Boripat Siriaroonrat 2

1 Department of Conservation, Research and Animals Health, Khao Kheow Open Zoo, Chonburi Province, Thailand 2 Bureau of Research, Conservation and Education, Zoological Park Organization, Thailand

3 Wildlife Clinic, Kasetsart University Veterinary Teaching Hospital, Kampaengsaen Campus, Nakornprathom Province.

1.Introduction The Eld�s deer has experienced the most severe decline in numbers during the last 60 years. The greatest threat to the extinction in the wild of Siamensis and Thamin Eld�s deers especially in Thailand continues to be poaching and habitat losses. In several Zoos and Wildlife sanctuary has a generally valuable female in a management breeding program but not given birth. Unlike in some population group, the reproductive rate of captive wildlife is highly variable until over population and inbreeding, because not enough of data to supporting or determine reproductive status which important for breeding program. A high priority is understanding the reproductive status of the captive population. Hormones drive the reproductive process, and endocrine patterns can indicate reproductive health (Brown et al., 2001). Remarkable progress has been made in assessing ovarian function in female, primarily through the analysis of excreted progestogen matabolites (Schwarzenberger et al., 1998). By Using non-invasive hormone monitoring by collecting fecal sampling. It is will provide valuable information on each female (Plame, 2005) because not all species or individuals permit the required level of handling. Fecal samples should be collected to the planned administration of the contraceptive implant to provide valuable reproductive status (ovarian activity, ovulation, pregnancy diagnosis, contraceptive assessment), assay validation and customized advice for breeding and research programs.(Morrow, 2007)

2. Objectives 2.1 Evaluating progesterone patterns in Females Rucervus eldi siamensis and Rucervus eldi thamin by Fecal steroids

extract 2.2 To determine individual reproductive physiological status of each animal, in order to use as a tool to evaluate

assisted reproductive technologies to be appropriate. 2.3 To rule out on hormonal database for cage management and success breeding in population group of Eld�s deer.

3. Methods 3.1 Animals and Fecal Sample Collection

Animals in this case study included 2 female Siamensis Eld�s deer (Rucervus eldii siamensis) and 8 female Thamin Eld�s deers at 3 institutions (2 zoos and 1 university). All animals were of adult age. Both species were put for data base

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requirement to breeding management plan by assisted reproductive technologies such as artificial insemination (AI) and embryo transfer (ET)

Individual fecal samples 10-50 g were collected one to three times weekly for 6- to 10- month periods. Criterions for fecal collection are fresh, no material contamination, and homogeneous feces so the metabolite is eventually distributed in fecal sample. After collections, samples were frozen and stored at -20 °C until analysis.

3.2 Fecal Steroid Extraction Frozen feces were dried in hot air oven at 60 °C until no change in weight of the sample is not detected, pulverized,

and the fecal powder was stored at -20°C until steroid extraction. A 0.1-g aliquot of well-mixed powder was boiled twice in 5 mL of 90 % ethanol for 20 minutes. After centrifuging at 2,500 g for 20 minutes, supernatants were collected, sediment were dissolved in 5 mL of 90 % ethanol. Extractants were vortexed (30 seconds) after that bring up centrifuged at 2,500 g for 15 minutes, supernatants were collected and combined in same tube before that, supernatants were recovered, dried, and redissolved in 1 mL dilution buffer. Extractans were vertexed (1 minute) and then stored at -20°C until steroid assays.

3.3 Assay Protocols Hormonal assay is performed by Competitive ELISA (Enzyme Linked Immuno Sorbent Assay) according to Brown et al.(2004). The progesterone EIA relied on a Monoclonal Antibody: Pregnane CL-425 (Provided by Quidel Corporation), progesterone standards (known concentrations), unknown (samples) concentrations of hormone (unlabeled antigen) and the hormone-specific enzyme conjugate (HRP) (the labeled antigen) are added to the well. The labeled and unlabeled antigens compete for binding sites on the antibody during the incubation phase. The substrate is added and reacts with the bound enzyme conjugate and changes color. The product of the substrate catalysis by enzyme is measured by transmitting light of a specific wavelength (405 nm.) through the product and measuring the amount of light adsorbtion by a plate reader (spectrophotometer). 3.4 Data analysis

Mean data are presented as ±SEM. Definition of the estrous cycle was based on fecal progestogen profiles. For each female, a nonpregnant baseline progestogen value was calculated using an iterative process in which values that exceeded the mean plus 1.5 standard deviations (SD) were excluded. The average was then recalculated and the elimination process was repeated until no values exceeded the mean plus 1.5 SD [Brown et al., 1994, 2001]

Onset of the luteal phase was defined as the first point after values increased above the baseline by 50 % and remained elevated for at least 2 consecutive weeks. The end of the luteal phase was defined as the first of two consecutive values that returned to baseline concentrations. Estrous cycle length was calculated as the beginning of the one luteal phase to the beginning of the next (Brown et al., 1994).

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4. Result and Conclusions Fecal progesterone assay has been used as noninvasive mean for assessing ovarian cyclicity in several species. However, progesterone pattern in Siamensis Eld�s deer (Rucervus eldii siamensis) has never been reported. The objective of the study was to assess progesterone pattern of Siamensis and Thamin Eld�s deers (Rucervus eldii thamin). Progesterone profile was detected in Siamensis (n=2) and Thamin Eld�s deers (n=8) by Enzyme immunoassay (EIA). The average estrous cycle length of Siamensis (n=11 cycles) and Thamin Eld�s deers (n=20 cycles) were 21.64±1.83 and 20.75±1.13 days, in range 12-32 and 14-30 days, respectively. In follicular phase, overall baseline of progesterone concentrations in Simensis and Thamin Eld�s deers were 298.91±11.99 and 172.0±28.09 ng/g, respectively. In luteal phase, progesterone concentrations in Simensis and Thamin Eld�s deers were ranging from 448 to 3,112 and 258.96 to 1,840 ng/g. Progesterone concentration of three pregnant Thamin Eld�s deers; one to term gestation (34 weeks after artificial insemination) and two premature births (32 weeks after embryo transfer) was investigated. During the first several weeks, progesterone concentrations remained at 400-1,000 ng/g and increased markedly during mid pregnancy (especially at 23rd week) with a peak concentrations reaching to 14,720 ng/g. Progesterone concentration in these Eld�s deers abrupt decline coincided with parturition (Fig 1). A similar gestation in Thamin Eld�s deers has been reported by Monfort et al.(1990), Hosack et al.(1997) and Morrow (2007). In conclusion, the study demonstrated the first report of Siamensis Eld�s deer progesterone profile (Fig.2a, b) compared to that of Thamin Eld�s deer. Fecal progesterone analysis would be useful for determining reproductive status (ovarian activity, estrous cycle, baseline concentrations and pregnancy diagnosis). Progesterone data base of individual Eld�s deer is important for breeding management by assisted reproductive technologies to be appropriate.

Fig 1. Fecal progesterone concentrations in an individual Eld�s Deer female (Rucervus eldi thamin)

throughout gestation.

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Fig. 2a, b. Individual profiles of fecal progesterone concentrations in two representative Eld�s Deer females (Rucervus eldi siamensis). Reproductive cycles are designated by an asterisk.

5. References Brown, J.L., Wasser, S.K., Wildt, D.E. and Graham, L.H. 1994. Comparative aspects of steroid hormone metabolism and

ovarian activity in felids, measured non-invasively in feces. Biol Reprod. (51): 776-786. Brown, J.L., Bellem, A.C., Michael, F., Wildt, D.E. and Roth, T.L. 2001. Comparative Analysis of Gonadal and Adrenal

Activity in The Black And White Rhinoceros in North America by Noninvasive Endocrine Monitoring. Zoo Biology. (20): 483-486.

Brown, J.L., Walker, S. and Steinman, K. 2004. Endocrine manual for the reproductive assessment of domestic and non-domestic species, 2nd edition, Smithsonian institution. USA.

Hosack, D.A., Miller, K.V., Marchinton, R.L. and Monfort, S.L. 1997. Ovarian Activity in Captive Eld�s Deer (Cervus eldi

thamin). Journal of Mammalogy. 78(2): 669-674.

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Monfort. S.L., Wemmer, C.M., Brown, J.L. and Wildt, D.E. 1990. Use of urinary hormone assays for evaluating endocrine

patterns associated with the long-day breeding season in Eld�s deer (Cervus eldi thamin). J. Exp. Zoo Suppl., (4): 215-8.

Morrow, C. 2007. FAQ - Frequently Asked Questions. [online]. Available from: http://scimitarscientific.com. [2011, March 15].

Palme, R. 2005. Measuring Fecal Steroids Guidelines for Practical Application. Academy of science, New York. Schwarzenberger, F., Walzer, C., Tomasova, K., Vahala, J., Meister, J., Goodrowe, K.L., Zima, J., Strauß, G. and Lynch, M.

1998. Fecal progesterone metabolite analysis for non-invasive monitoring of reproductive function in the white

rhinoceros (Ceratotherium simum). Anim Reprod Sci, (53): 173-90.

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Assessment of Breeding Difficulties in Captive Black-necked Stork (Ephippiorhynchus asiaticus) by Hormonal Study at Khao Kheow Open Zoo,Thailand

Urarikha Kongprom1, Chainarong Punkong1, Pinanong Thongnoppakun1, Nittaya Petsukorn1, Shongkrod Poothong 2, and Rattapan Pattanarangsan3

1 Department of Conservation, Research and Animals Health, Khao Kheow Open Zoo, Chonburi Province, Thailand 2 Brangpra Waterfowl Breeding Station, Wildlife sanctuary, Chonburi Province.

3 Veterinary, Mahidol University, Nakornprathom Province.

1.Introduction The Black-necked stork (Ephippiorhynchus asiaticus) is one of the tallest extant species of storks. Over the past 30

years, it originates from the Wet land in Southeast Asia (Except in Malaysia, Indonesia and Philippine), and partly of South Asia and also Australia. As the Black-necked stork is threatened in the wild, especially in several country of Southeast Asia such as Thailand. The greatest threat to the extinction in the wild of Thailand continues to be poaching and habitat losses from the fast development of social industry, it is important that a viable population is maintained in captivity. Before that, it is one main objective for The Zoological Park Organization of Thailand, under the royal patronage to conduct conservation breeding. This will help ex situ conservation, and also biodiversity conservation. Black-necked Storks are in our focus of this objective. However, breeding in captivity is relatively erratic. There are many factors that involving with unsuccessful breeding in captivity such as, infertility and non-paired breeding. In the present study describes a project initialed to establish basic reproductive parameters in this species, the diagnosis and monitoring of hormonal are two of the most important reproductive evaluation that can be made for the management of animals in breeding programs, with the ultimate aim of achieving better breeding success. Hormonal detection permits initiation of monitoring and health care for the animal individual. Knowledge of the reproductive status of each female allows animals managers to optimize production of a colony to ensure sustainable conservation of endangered species.

Enzyme immunoassays (EIAs) have been developed for evaluation of hormone metabolites in nondomestic species. These EIAs have the advantages of being less expensive to perform and do not require the use of controlled radioisotopes (Czekala et al., 1986) Non-invasive methods using in quantification of steroid hormones in feces is one suitable way for captive wild animals (Rupert et al., 2005).This method can avoid traumatizing or stressing the animals from capture and can be explained in the principal of hormone metabolism. Hormones produced from endocrine glands or tissues will be excreted into blood stream to the target organs or tissues. Then, the hormones will pass through liver, where they were change to different metabolites. Metabolites then excreted into bile system and finally the bile with metabolites is mixed with feces and expelled out of the body. Metabolite levels can reflect Steroid hormones level. All the detectable steroids in feces are called �Fecal steroids� (Erich et al., 2005)

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2. Objectives 2.1 To quantify levels of stress hormone in Black necked stork from fecal extraction method in order to use as a tool to

evaluate cage management 2.2 To determine individual reproductive physiological status of each animal in different reproductive stages in captive

male and females. 2.3 To quantify levels of nutrition requirement in Black neck storks on average years round

3. Methods 3.1 Materials (Essential Component of the EIA)

• Solid Phase: polystyrene microtiter plate (Nunc Immuno plate) • Antibody: Polyclonal Antibody and Monoclonal Antibody • Coating buffer: Carbonate/bicarbonate buffer pH 9.6 • Wash solution: Nacl and Tween 20 solution • Enzyme conjugate (tracer): Horseradish peroxidase (HRP) • Assay buffer: Phosphate or Tris buffers pH 7.0 • Standards: Known concentrations of hormone (Sigma Diagnostics) • Substate: Chromagen (ABTS) and catalyst (Hydrogen peroxide) Substrate • Reading: Spectrophotometer or Plate reader

3.2 Collection of Storks dropping: Individual fecal samples 10-50 g were collected one to three times weekly for 12 to 24- month periods. Criterions for fecal collection are fresh, no material contamination, and homogeneous feces so the metabolite is eventually distributed in fecal sample. After collections, samples were frozen and stored at -20 °C until analysis. 3.3 Preservation with Freezing or Drying immediately: Incase of drying process is delayed, we collect fecal sample in dark, close container to prevent from moisture and light. Then the box is kept at -20 degree Celsius, in order to prevent the sample from bacterial activities which can interfere with steroid levels Drying of sample is done by lyophilizer or hot air oven until no change in weight of the sample is not detected. Hot air oven is set at 96 hours, at 60 degree Celsius. Dry sample is collected in close container with moisture absorbent substance, in the refrigerator. (Erich et al., 2005; Tony et al., 2005) 3.4 Fecal Extraction: Extraction process is done by boiling 0.2 g of dry sample in 90% Alcohol. Then the sample is kept at -20 degree Celsius for the analytical process

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3.5 Assay Protocols: Hormonal assay is performed by Competitive ELISA (Enzyme Linked Immuno Sorbent Assay) according to Brown et al (2004) ELISA or Enzyme-linked Immuno Sorbent Assay works on the principals of reaction between coated specific antibody, to the particular substance, and antigen in the sample solution. The plate is coated with antibody which will adhere with the antigen from the sample and also from the conjugated enzyme. Both type of antigen will compete for coated antibody on the plate. Then free antigen of both antigens will be washed out. The remaining antigen with enzyme will change substrate in gradient levels according to amount of enzyme attached on antigen and antibody. Quantification can be done by comparing with color produced by known amount of antigen, by photometer machine. 3.6 Data analysis

Mean data are presented as ±SEM. Definition of the reproductive status was based on fecal hormone profiles. For each individual, a non pregnant baseline progesterone value was calculated using an iterative process in which values that exceeded the mean plus 1.5 standard deviations (SD) were excluded. The average was then recalculated and the elimination process was repeated until no values exceeded the mean plus 1.5 SD [Brown et al., 1994, 2001]

4. Result and Conclusions The results of the Assessment of Breeding Difficulties in Captive Black-necked Stork (Ephippiorhynchus asiaticus)

by Hormonal Study: we group animals into various groups for comparisons as;

Black-necked Stork females All study females showed at least some evidence of ovarian activity. There was seasonality in ovarian activity because cycles were not observed every month of years, with observed only in Breeding season (October-November or January-February). Anestrous periods, ranging from as long as 8 to 10 consecutive months. One singleton female has successful to breed and given birth during a 2-year period. Others not success to breeding pair or not given birth, were observed in 3 females; however, one of these episodes occurred sickness and died in later. There was no age difference (P>0.05) between paired and non-paired females in this study population. The progesterone concentrations in breeding season of all females were significantly different between paired female and non-paired females, with paired females had higher than two non-paired females (P<0.05) but non-significant different in another time with non-breed season during a year period (P>0.05) (Fig. 1). For each female, a non-breeding (out of breeding time) Overall baseline concentrations of fecal progesterone averaged 77.8 ± 1.91 ng/g of dry feces, with peak concentrations ranging from approximately 12 to 180 ng/g

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Fig.1. Fecal progesterone concentrations in an individual Black-necked Stork female

throughout average one year round.

Fecal total 17-ß Estradiol metabolite concentrations in Black-necked stork females fluctuated markedly and were not useful for characterizing follicular activity, even when samples were collected three times per week (Fig. 2). All those compared mean average 17-ß Estradiol concentration in female between breeding paired and non-paired female group, there was non-significant different in paired and non-paired (non-breeding) group (P > 0.05), however 17-ß Estradiol level in paired female to be seem higher than in non-paired females. For fecal cortisol concentrations, when compare the overall cortisol concentrations of all females were significantly different between paired female and non-paired females, with paired female had lower than two non-paired females (P < 0.05) were 17.58±1.04 ng/g and 23.57±1.03 ng/g of dry feces respectively. Finally, in paired female at Khao Kheow Open Zoo can bred 2 times and lay several of eggs in single season from cage management.

Fig.2. Fecal 17-ß Estradiol concentrations in an individual Black-necked Stork females throughout average one year round.

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Black-necked Stork males The results of the Testosterone in Black-necked stork males are depicted in Figures 3 a, b. In contrast to females, there was difference (P<0.05) between paired male and three non-paired males in this study population. The average age of paired male was greater (P<0.05) than that for the non-paired males. There was no evidence of seasonality in testicular activity. Overall mean fecal testosterone concentrations were higher (P<0.05) in the paired male (205.60±13.10 ng/g of dry feces); range, 21-877 ng/g) than in non-paired males (128.71±4.76 ng/g of dry feces); range, 8-1,042 ng/g). All this when compared in the same non-breeding group, there was no difference (P>0.05) in average fecal testosterone concentrations. The study also found that cortisol level was highest in breeding season for paired male. Another male was highest in the summer.

Fig. 3 a, b. Fecal Testosterone concentrations in an individual Black-necked Stork males throughout average one year round.

5. References Brown, J.L., Wasser, S.K., Wildt, D.E. and Graham, L.H. 1994. Comparative aspects of steroid hormone metabolism and

ovarian activity in felids, measured non-invasively in feces. Biol Reprod. 51: 776-786. Brown, J.L., Bellem, A.C., Michael, F., Wildt, D.E. and Roth, T.L. 2001. Comparative Analysis of Gonadal and Adrenal

Activity in The Black And White Rhinoceros in North America by Noninvasive Endocrine Monitoring. Zoo Biology. 20: 483-486.

Brown, J.L., Walker, S. and Steinman, K. 2004. Endocrine manual for the reproductive assessment of domestic and non-domestic species, 2nd edition, Smithsonian institution. USA.

Czekala, N.M., Gallusser, S., Meier, M.E., Lasley, B.L. 1986. The development and application of an enzyme immunoassay for urinary estrone conjugates. Zoo Biology. 5: 1-16.

Erich Mostl, Sophi Rettenbacher, And Rupert Palme. 2005. Measurement of Corticosterone Metabolites in Birds� Droppings: An Analytical Approach. Ann. N.Y. Acad. Sci. 1046: 17 34.

Palme, R. 2005. Measuring Fecal Steroids Guidelines for Practical Application. Academy of science, New York.

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Toni E. Ziegler, and Daniel J. Wittwer. 2005. Fecal Steroid Research in the Field and Laboratory Improved Methods for Storage, Transport, Processing, and Analysis. University of Wisconsin. American Journal of Primatology, 67 : 159-174.

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�KE�����AQ>�J�@G T��J�A> Mean ± SEM. �D�Dก�G���A> @B�BCก�@Y�C��P�?>�JK��CYS�u�A> �J���ก�E�������� �����o>�?R�� ��S ��S�KE�����u������o>�?R��T�@���?���]�K]GQ��S �Q> ��TNQ��P�ก��D���WSDV �� iterative process RG���G�K���Eก�� 1.5 E�KA> �KE���� E�����u� (1.5 SD) >>ก�Q��Dก��D���WSD]�E�Y�>�V ��]�K���KTGEก��ก�K 1.5 E�KA> �KE���� E�����u�>�ก [Brown et al., 1994, 2001]

4. _2ก��[\กU�12$��7#_2 �กc�ก���ก�ก����E����rUI>��@���T�ก�A��C��P�?�กก��@�>GD Black-necked Stork (Ephippiorhynchus

asiaticus) T�ก� E��S� RG���P���ก������o>�?R�� @��_@G c�E�f�ก��K�V ]GQG� ��S �กก�$��(0.V�%)[%&�5 �กก���ก������o>�?R��T�EC�E�����S I�G C��K��AQ>�K N�S���@G TIQEIe�_� ก��D �A> �� ]AK RG�C��K�� ]AKT�����@Y�C��P�?A> �กก��@�>GDEC�E�����ก��D �E�f�NK� XGJก�RG�@G _� ����ก�K T�� NK� EGY>�A> �>��� W�� @G ]GQT�NK� ���E�f�XGJก�@Y�C��P�? (EGY>������-CX���ก�� I�Y> �ก���-ก��BC��P?) ��S ��SC��KNK� ���E�f�����C�กI�Y> Anestrous ���������>�JKT�NK� 8 _� 10 EGY>��K>E�Y�> ก�� �กก���ก�T�EC�E�����S I�G 3 ��� ��EC�� ���EG���������@����@DE�e�T�ก�����JK��TIQ�Jก]GQ��>GNK� ����E�� 2 ��A> ก���ก� @K�����EI�Y>]�K���@����@DE�e�T�ก�����JKA��C��P�? (RG���EC�E����D��� 1 ��� �=������T�E���K>�) �กก���ก�]�KC�����ก�K (P > 0.05) A> NK�

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>����I�K EC�E����������JK]GQ������JK]�K]GQ (paired and non-paired females) ��S ��SC��K�����o>�?R��R��E�@E�>R��T�NK� ���E�f�XGJก�@Y�C��P�?A> �กก��@�>GD��I�K EC�E����������JKA��C��P�?]GQก��ก��K����]�K����JK ������ก�K ก��>�K �����@D��U� @_��� (P < 0.05) RG�EC�E����������JK]GQ����CX��ก���ก�A��C��P�?������������EAQ�AQ�A> o>�?R��R��E�@E�>R��@J ก�KEC�E���>�ก 2 ��� W�� E�f�ก��K����]�K����JK @DI���T�NK� EGY>�>Y��V A> ��C��K��G��A> �����o>�?R��������ก�K >�K ]�K�����@D��U� @_��� (P>0.05) (BC��� 1) @DI���ก���ก��KE�����CYS�u�A> o>�?R��R��E�@E�>R�� (baseline concentrations of fecal progesterone) A> EC�E�����S I�G (]�K������NK� E�������ก�@Y�C��P�?) C��K�กก��@�>GDEC�E������KE�����CYS�u�A> oo�?R��R��E�@E�>R��E�����E�Kก�� 77.8 ± 1.91 ng/g of dry feces, C�@�� 12 - 180 ng/g

��)���.1. Fecal progesterone concentrations in an individual Black-necked Stork female throughout average one year round.

@DI���ก�������G�����o>�?R�� 17 -ß E>@��]G>>� E����>]�E�@ (17-ß Estradiol metabolite) T�

�กก��@�>GDEC�E��� �กc�ก���ก�C��K��c�A> o>�?R��G� ก�K��K>�AQ �����������@J ��ก�A�S���� @���ก�� �� >�]�K@��_�D�TNQ@G I�Y>N�S�K NK� A> ก��ก]AK]GQ>�K N�GE���ก��กT�ก���ก������ก�Eกe����>�K �� 3 ���S �K>@��GI? (BC��� 2.) W�� E�Y�>E�����E������G�����EAQ�AQ�E�����A> o>�?R�� 17-ß Estradiol ��I�K EC�E����������JKA��C��P�?]GQก��ก��K��������JK]�K]GQC��K������ก�K ก��>�K ]�K�����@D��U� @_��� (P > 0.05) ��S ��ST�EC�E����������JKA��C��P�?]GQ����G�����EAQ�AQ�E�����A> o>�?R�� 17-ß Estradiol @J ก�Kก��K�EC�E����������JK]�K]GQE�eก�Q>� @DI���ก�������G��G��A> o>�?R���>�?��W>� (fecal cortisol) E�Y�>�Dก�E�����E������I�K EC�E����������JK]GQก��ก��K��������JK]�K]GQ C��K������ก�K ก��>�K �����@D��U� @_���(P < 0.05) RG�EC�E����������JK]GQ����G�����EAQ�AQ�A> �>�?��W>���Dก�Kก��K����]�K]GQ����JK W�� ����G�����EAQ�AQ�E�����E�Kก�� 17.58±1.04 ng/g �� 23.57±1.03 ng/g of dry feces ���DG�� �>ก�ก��S�กก���ก�C��K�กก��@�>GD�JKCK>�KC��P�?A> @��@���?E�FGEAEA���@��_�D�� � ]AK]GQ 2 ���S �K>�� �กc�ก���Gก�ก� E��S� A> @��@���?v

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��)���.2. Fecal 17-ß Estradiol concentrations in an individual Black-necked Stork females throughout average one year round.

�กก�$��(0.V�%)[_9/ T�ก���ก�R�� @�Q >��A> �กก��@�>GDEC�cJQ�D��� 4 ��� C��KEC�cJQ�������JK��A��C��P�?]GQ����>���กก�KEC�cJQ>�ก 3 ����������JK]�K]GQ>�K �����@D��U� @_��� (P<0.05) W�� �ก�K ก�����C�T�EC�E��� ��S ��ST�ก�������G�����o>�?R��EC�cJQ�Y>o>�?R��E�@R�@E�>R�� (Fecal Testosterone) ���@��_@G ]GQG� BC��� 3 a, b C��K��R�Q�RG�E�����A> �����o>�?R��]�K@G _� ก�E�������� ���E�f�NK� XGJก� (seasonality in testicular activity) �KT�EC�cJQ�������JK�Q�������G��A> o>�?R��E�@R�@E�>R��@J ก�KEC�cJQT�ก��K������ ����JK]�K]GQ����>���Q>�ก�K>�K �����@D��U (P<0.05) RG�����G�����EAQ�AQ�A> o>�?R��E�@R�@E�>R��E�������S I�GT��>���E�Kก�� 205.60±13.10 ng/g of dry feces (C�@�� 21-877 ng/g) ��128.71±4.76 ng/g of dry feces (C�@�� 8-1,042 ng/g) ���DG�� �KE�Y�>E�����E����E�C�T�ก��K�A> EC�cJQ���]�K����JK�D��� 3 ���C��K������ก�K A> �����o>�?R��>�K ]�K�����@D��U� @_��� (P>0.05)

�>ก�ก��S�� C��KT��กก��@�>GDEC�cJQ���]�K]GQ����JK������G��A> o>�?R�����E����G (stress hormone) I�Y>o>�?R���>�?��W>� (Fecal Cortisol) @J @�GT�NK� XGJ�Q>� @K��EC�cJQ�������JK�Q�������G��A> o>�?R���>�?��W>� (Fecal Cortisol) @J @�GT�NK� ���E�f�XGJก�@Y�C��P�?

��)��� 3 a, b. Fecal Testosterone concentrations in an individual Black-necked Stork males

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throughout average one year round.

5. %0ก���0/�,0', Brown, J.L., Wasser, S.K., Wildt, D.E. and Graham, L.H. 1994. Comparative aspects of steroid hormone metabolism and

ovarian activity in felids, measured non-invasively in feces. Biol Reprod. 51: 776-786. Brown, J.L., Bellem, A.C., Michael, F., Wildt, D.E. and Roth, T.L. 2001. Comparative Analysis of Gonadal and Adrenal

Activity in The Black And White Rhinoceros in North America by Noninvasive Endocrine Monitoring. Zoo Biology. 20: 483-486.

Brown, J.L., Walker, S. and Steinman, K. 2004. Endocrine manual for the reproductive assessment of domestic and non-domestic species, 2nd edition, Smithsonian institution. USA.

Czekala, N.M., Gallusser, S., Meier, M.E., Lasley, B.L. 1986. The development and application of an enzyme immunoassay for urinary estrone conjugates. Zoo Biology. 5: 1-16.

Erich Mostl, Sophi Rettenbacher, And Rupert Palme. 2005. Measurement of Corticosterone Metabolites in Birds� Droppings: An Analytical Approach. Ann. N.Y. Acad. Sci. 1046: 17 34.

Palme, R. 2005. Measuring Fecal Steroids Guidelines for Practical Application. Academy of science, New York. Toni E. Ziegler, and Daniel J. Wittwer. 2005. Fecal Steroid Research in the Field and Laboratory Improved Methods for

Storage, Transport, Processing, and Analysis. University of Wisconsin. American Journal of Primatology, 67 : 159-174.

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ก��[\กU�%#��53%��53�$.�3j0���&�(0���'h028�+&�(��5%)[_9/ (Ursus thibetanus thibetanus) �$+���,)`4��������1�.,12$[9�5��,%(��$+������60,��������%#;.%6�%6�5�

Comparative study of Cortisol metabolites level in Male Asiatic Black Bears

(Ursus thibetanus thibetanus) between exhibited enclosure and surplus enclosure at Khao Kheow Open Zoo .

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3�(�.5�0 �กก���ก�ก�������G @E����>�G? Glucocorticoids Metabolized T����>�K �J�A> I�����ECY�>E�����E������G��

o>�?R���>�?��W>���I�K 2 ก��K����Nก��Y> ก��K���G �ก� 95 ����G@K��@G �K>I�� �����ก���Gก�ก� E��S� ����ก�K ก�� GQ����P�ก�������E���I?�� Enzyme immunoassay C��KI�������S @> ก��K�����������EAQ�AQ�A> o>�?R���>�?��W>��ก�K ก�� RG�I��������>�JKT����E����G ��K>I���������o>�?R���>�?��W>�E�����E�Kก�� 24.34±19.94 ng/g (n = 40) @K��T�I��������>�JK���E����G �ก� 95 �������o>�?R��G� ก�K� E�����E�Kก�� 15.47±7.68 ng/g (n = 40) W�� E�Y�>E�����E���������o>�?R���>�?��W>�E�������I�K @> ก��K����Nก�C��KI��������>�JK���E����G ��K>I���������o>�?R��G� ก�K�E�����@J ก�K���C�T���G �ก� 95 (P>0.05, n = 80) ��E�Y�>C����T�BC���C��KI�����A> @��@���?E�FGEAEA�����S @> ก��K��������o>�?R���>�?��W>����E�f��KE�����CYS�u� (Average baseline concentrations) E�Kก�� (Mean±SEM) 11.15 ± 0.82 ng/g of dry, C�@�� (3.83 -65.37 ng/g)

��S ��SE�Y�>C����E�����E���������o>�?R���>�?��W>�E�C�T�ก��K����Nก�EG���ก�� C��KB�T���G ���]�K�����@D��U (P>0.05) ��I�K I������K����� ENK�EG���ก��T���S @> ก��K����Nก� ��@G TIQEIe�_� ก�]�K�����@D��U��I�K NK� >������ก�K ก��B�T�ก��K� (P>0.05) �>ก�ก��SE�Y�>C�����J���A> ก���Gก�ก� E��S� � GQ��K V �K��ก��ก��ก���ก������o>�?R��C��K ก���Gก�ก� E��S� ����ก�K ก����S T�E�Y�> A> ก�E��S� �����ก��K� @BC�G�Q>�B�T�ก� , A�Gก� , >I���NK� E��ก�TIQ>I���c��K>CX��ก���@���?W�� ��c��K>ก�I��� �����o>�?R���>�?��W>�����ก�K ก��RG�����R�Q��Kก��K����Nก����@K E@���TIQ@���?]GQ��ก��ก���T�ก�GDE���N��������D�������ก�@G >>ก� CX��ก����กก�K ����ก�I��� o>�?R���>�?��W>�T����������กก�KGQ�� ��S ��SC��Kก���ก�������>�?��W>�@��_�����TNQ�D�@G _� ����ก�K A> ก���Gก�ก� E��S� ]GQ ��@��_�DAQ>�J����]GQ]�TNQกQ]A�rUIT�ก���Gก�@���?�=]GQT���G��I��� >�ก��S �� C��Kก�TNQ������>�?��W>�E�f�G�N��N�S��GB�����E����GT�@���?�Q> TNQ����J]�ก��AQ>�J����Eก����AQ> ก��ก���Gก�ก� E��S� �K��GQ���� ��]GQAQ>�J����_Jก�Q> �ก��� A�S�. (V��V�(�:: Enzyme immunoassay, Glucocorticoids Metabolized, �>�?��W>�, ก���Gก�ก� E��S� ,

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Abstract

The noninvasive methods to measure fecal steroids hormone (Glucocorticoids Metabolized) level in feces of Male

Asiatic Black Bears for comparative study of Cortisols\levels between exhibited enclosure and surplus enclosure (individual separately groups) with different captive management by Enzyme immunoassay, the reported levels are four males averages. A one-way ANOVA showed different data in average cortisol level between groups. Before that From exhibited enclosure group, mean average cortisol level is equal to 24.34±19.94 ng/g of dry feces (n = 40), males of captive housed separately group, mean average cortisol level is equal to 15.47 ± 7.68 ng/g of dry feces (n = 40). When compared of average cortisol level between two groups, there was no significant different in average cortisol concentration among exhibited enclosure and captive housed separately groups during the study (P > 0.05, n = 80). However cortisol level in exhibited enclosure to be seem higher than in captive housed separately groups. For overall baseline concentrations of fecal cortisol averaged 11.15 ± 0.82 ng/g of dry feces, ranging from approximately 3.83 to 65.37 ng/g

When compared in the same breeding group, there was no significant different in average cortisol concentration between individuals (P > 0.05), similarly in two groups. Males of both group showed no evidence of age on the basis of cortisol metabolites concentrations too. In summary, fecal steroid analysis identified a number of differences in Cortisol hormonal secretory dynamics between Exhibited enclosure and Captive housed separately group that may be related to difference in captive management as well as inner environment breeding cages, cage�s dimensions, food diversity and time of feeding in captivity. Most Asiatic black bear males exhibited demonstrated evidence of regular cortisol metabolites level, and those males had some active activity were higher than in captive housed separately. Results also suggest that fecal corticoid concentrations can be useful for understanding the difference efficiency of cage management and cause of inconsistent behavior to healthy in wildlife. However, continued studies in stress are needed to consideration together with data from captive management to be appropriate.

Key word: Enzyme immunoassay, Glucocorticoids Metabolized, Cortisol, Captive management

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