����JFIFXX�����    $.' ",#(7),01444'9=82<.342  2!!22222222222222222222222222222222222222222222222222����"��4�� ���,�PG"Z_�4�˷����kjز�Z�,F+��_z�,�© �����zh6�٨�ic�fu���#ډb���_�N�?��wQ���5-�~�I���8����TK<5o�Iv-�����k�_U_�����~b�M��d����Ӝ�U�Hh��?]��E�w��Q���k�{��_}qFW7HTՑ��Y��F�?_�'ϔ��_�Ջt��=||I ��6�έ"�����D���/[�k�9���Y�8ds|\���Ҿp6�Ҵ���]��.����6�z<�v��@]�i%��$j��~�g��J>��no����pM[me�i$[����s�o�ᘨ�˸ nɜG-�ĨU�ycP�3.DB�li�;��hj���x7Z^�N�h������N3u{�:j�x�힞��#M&��jL P@_���� P��&��o8������9�����@Sz6�t7#O�ߋ �s}Yf�T���lmr����Z)'N��k�۞p����w\�Tȯ?�8`�O��i{wﭹW�[�r�� ��Q4F�׊���3m&L�=��h3����z~��#�\�l :�F,j@�� ʱ�wQT����8�"kJO���6�֚l����}���R�>ډK���]��y����&����p�}b��;N�1�m�r$�|��7�>e�@B�TM*-iH��g�D�)� E�m�|�ؘbҗ�a��Ҿ����t4���o���G��*oCN�rP���Q��@z,|?W[0�����:�n,jWiE��W��$~/�hp\��?��{(�0���+�Y8rΟ�+����>S-S����VN;�}�s?.����� w�9��˟<���Mq4�Wv'��{)0�1mB��V����W[�����8�/<� �%���wT^�5���b��)iM� pg�N�&ݝ��VO~�q���u���9� ����!��J27����$O-���! �:�%H��� ـ����y�ΠM=t{!S�� oK8������t<����è:a������[�����ա�H���~��w��Qz`�po�^ ����Q��n� �,uu�C�$ ^���,������8�#��:�6��e�|~���!�3�3.�\0��q��o�4`.|� ����y�Q�`~;�d�ׯ,��O�Zw�������`73�v�܋�<���Ȏ�� ـ4k��5�K�a�u�=9Yd��$>x�A�&�� j0� ���vF��� Y�|�y��� ~�6�@c��1vOp�Ig����4��l�OD���L����� R���c���j�_�uX6��3?nk��Wy�f;^*B� ��@�~a�`��Eu������+���6�L��.ü>��}y���}_�O�6�͐�:�YrG�X��kG�����l^w���~㒶sy��Iu�!� W ��X��N�7BV��O��!X�2����wvG�R�f�T#�����t�/?���%8�^�W�aT��G�cL�M���I��(J����1~�8�?aT ���]����AS�E��(��*E}� 2��#I/�׍qz��^t�̔���b�Yz4x���t�){ OH��+(E��A&�N�������XT��o��"�XC��'���)}�J�z�p� ��~5�}�^����+�6����w��c��Q�|Lp�d�H��}�(�.|����k��c4^�"�����Z?ȕ ��a<�L�!039C� �Eu�C�F�Ew�ç ;�n?�*o���B�8�bʝ���'#Rqf���M}7����]����s2tcS{�\icTx;�\��7K���P���ʇ Z O-��~��c>"��?�������P��E��O�8��@�8��G��Q�g�a�Վ���󁶠�䧘��_%#r�>�1�z�a��eb��qcPѵ��n���#L��� =��׀t� L�7�`��V���A{�C:�g���e@�w1 Xp3�c3�ġ����p��M"'-�@n4���fG��B3�DJ�8[Jo�ߐ���gK)ƛ��$���� ���8�3�����+���� �����6�ʻ���� ���S�kI�*KZlT _`���?��K����QK�d����B`�s}�>���`��*�>��,*@J�d�oF*����弝��O}�k��s��]��y�ߘ��c1G�V���<=�7��7����6�q�PT��tXԀ�!9*4�4Tހ3XΛex�46���Y��D ����� �BdemDa����\�_l,��G�/���֌7���Y�](�xTt^%�GE�����4�}bT���ڹ�����;Y)���B�Q��u��>J/J �⮶.�XԄ��j�ݳ�+E��d ��r�5�_D�1 ��o�� �B�x�΢�#���<��W�����8���R6�@g�M�.��� dr�D��>(otU��@x=��~v���2� ӣ�d�oBd��3�eO�6�㣷�����ݜ6��6Y��Qz`��S��{���\P�~z m5{J/L��1������<�e�ͅPu�b�]�ϔ���'������f�b� Zpw��c`"��i���BD@:)ִ�:�]��hv�E�w���T�l��P���"Ju�}��وV J��G6��. J/�Qgl߭�e�����@�z�Zev2u�)]կ�����7x���s�M�-<ɯ�c��r�v�����@��$�ޮ}lk���a���'����>x��O\�ZFu>�����ck#��&:��`�$�ai�>2Δ����l���oF[h��lE�ܺ�Πk:)���`�� $[6�����9�����kOw�\|���8}������ބ:��񶐕��I�A1/�=�2[�,�!��.}gN#�u����b��� ~��݊��}34q����d�E��Lc��$��"�[q�U�硬g^��%B �z���r�pJ�ru%v\h1Y�ne`ǥ:g���pQM~�^�Xi� ��`S�:V29.�P���V�?B�k�� AEvw%�_�9C�Q����wKekPؠ�\�;Io d�{ ߞo�c1eP����\� `����E=���@K<�Y���eڼ�J���w����{av�F�'�M�@/J��+9p���|]�����Iw &`��8���&M�hg��[�{��Xj��%��Ӓ�$��(����ʹN���<>�I���RY���K2�NPlL�ɀ)��&e����B+ь����( � �JTx���_?EZ� }@ 6�U���뙢ط�z��dWI�n` D����噥�[��uV��"�G&Ú����2g�}&m��?ċ�"����Om#��������� ��{�ON��"S�X��Ne��ysQ���@Fn��Vg���dX�~nj�]J�<�K]:��FW��b�������62�=��5f����JKw��bf�X�55��~J �%^����:�-�QIE��P��v�nZum� z � ~ə ���� ���ة����;�f��\v���g�8�1��f24;�V���ǔ�)����9���1\��c��v�/'Ƞ�w�������$�4�R-��t���� e�6�/�ġ �̕Ecy�J���u�B���<�W�ַ~�w[B1L۲�-JS΂�{���΃������A��20�c#��@ 0!1@AP"#2Q`$3V�%45a6�FRUq��� ����^7ׅ,$n�������+��F�`��2X'��0vM��p�L=������5��8������u�p~���.�`r�����\���O��,ư�0oS ��_�M�����l���4�kv\JSd���x���SW�<��Ae�IX����������$I���w�:S���y���›R��9�Q[���,�5�;�@]�%���u�@ *ro�lbI �� ��+���%m:�͇ZV�����u�̉����θau<�fc�.����{�4Ա� �Q����*�Sm��8\ujqs]{kN���)qO�y�_*dJ�b�7���yQqI&9�ԌK!�M}�R�;������S�T���1���i[U�ɵz�]��U)V�S6���3$K{�ߊ<�(� E]Զ[ǼENg�����'�\?#)Dkf��J���o��v���'�%ƞ�&K�u�!��b�35LX�Ϸ��63$K�a�;�9>,R��W��3�3� d�JeTYE.Mϧ��-�o�j3+y��y^�c�������VO�9NV\nd�1 ��!͕_)a�v;����թ�M�lWR1��)El��P;��yوÏ�u 3�k�5Pr6<�⒲l�!˞*��u־�n�!�l:����UNW ��%��Chx8vL'��X�@��*��)���̮��ˍ��� ���D-M�+J�U�kvK����+�x8��cY������?�Ԡ��~3mo��|�u@[XeY�C�\Kp�x8�oC�C�&����N�~3-H���� ��MX�s�u<`���~"WL��$8ξ��3���a�)|:@�m�\���^�`�@ҷ)�5p+��6���p�%i)P M���ngc�����#0Aruz���RL+xSS?���ʮ}()#�t��mˇ!��0}}y����<�e� �-ή�Ԩ��X������ MF���ԙ~l L.3���}�V뽺�v�����멬��Nl�)�2����^�Iq��a��M��qG��T�����c3#������3U�Ǎ���}��לS�|qa��ڃ�+���-��2�f����/��bz��ڐ�� �ݼ[2�ç����k�X�2�* �Z�d���J�G����M*9W���s{��w���T��x��y,�in�O�v��]���n����P�$�JB@=4�OTI�n��e�22a\����q�d���%�$��(���:���: /*�K[PR�fr\nڙdN���F�n�$�4�[�� U�zƶ����� �mʋ���,�ao�u 3�z� �x��Kn����\[��VFmbE;�_U��&V�Gg�]L�۪&#n%�$ɯ�dG���D�TI=�%+AB�Ru#��b4�1�»x�cs�YzڙJG��f��Il��d�eF'T� iA��T���uC�$����Y��H?����[!G`}���ͪ� �纤Hv\������j�Ex�K���!���OiƸ�Yj�+u-<���'q����uN�*�r\��+�]���<�wOZ.fp�ێ��,-*)V?j-kÊ#�`�r��dV����(�ݽBk�����G�ƛk�QmUڗe��Z���f}|����8�8��a���i��3'J�����~G_�^���d�8w������ R�`(�~�.��u���l�s+g�bv���W���lGc}��u���afE~1�Ue������Z�0�8�=e�� f@/�jqEKQQ�J��oN��J���W5~M>$6�Lt�;$ʳ{���^��6�{����v6���ķܰg�V�cnn �~z�x�«�,2�u�?cE+Ș�H؎�%�Za�)���X>uW�Tz�Nyo����s���FQƤ��$��*�&�LLXL)�1�" L��eO��ɟ�9=���:t��Z���c��Ž���Y?�ӭV�wv�~,Y��r�ۗ�|�y��GaF�����C�����.�+� ���v1���fήJ�����]�S��T��B��n5sW}y�$��~z�'�c ��8 ��� ,! �p��VN�S��N�N�q��y8z˱�A��4��*��'������2n<�s���^ǧ˭P�Jޮɏ�U�G�L�J�*#��<�V��t7�8����TĜ>��i}K%,���)[��z�21z ?�N�i�n1?T�I�R#��m-�����������������1����lA�`��fT5+��ܐ�c�q՝��ʐ��,���3�f2U�եmab��#ŠdQ�y>\��)�SLY����w#��.���ʑ�f��� ,"+�w�~�N�'�c�O�3F�������N<���)j��&��,-� �љ���֊�_�zS���TǦ����w�>��?�������n��U仆�V���e�����0���$�C�d���rP �m�׈e�Xm�Vu� �L��.�bֹ��� �[Դaզ���*��\y�8�Է:�Ez\�0�Kq�C b��̘��cө���Q��=0Y��s�N��S.���3.���O�o:���#���v7�[#߫ ��5�܎�L���Er4���9n��COWlG�^��0k�%<���ZB���aB_���������'=��{i�v�l�$�uC���mƎҝ{�c㱼�y]���W�i ��ߧc��m�H� m�"�"�����;Y�ߝ�Z�Ǔ�����:S#��|}�y�,/k�Ld� TA�(�AI$+I3��;Y*���Z��}|��ӧO��d�v��..#:n��f>�>���ȶI�TX��� 8��y����"d�R�|�)0���=���n4��6ⲑ�+��r<�O�܂~zh�z����7ܓ�HH�Ga롏���nCo�>������a ���~]���R���̲c?�6(�q�;5%� |�uj�~z8R=X��I�V=�|{v�Gj\gc��q����z�؋%M�ߍ����1y��#��@f^���^�>N�����#x#۹��6�Y~�?�dfPO��{��P�4��V��u1E1J �*|���%���JN��`eWu�zk M6���q t[�� ��g�G���v��WIG��u_ft����5�j�"�Y�:T��ɐ���*�;� e5���4����q$C��2d�}���� _S�L#m�Yp��O�.�C�;��c����Hi#֩%+) �Ӎ��ƲV���SYź��g |���tj��3�8���r|���V��1#;.SQ�A[���S������#���`n�+���$��$I �P\[�@�s��(�ED�z���P��])8�G#��0B��[ى��X�II�q<��9�~[Z멜�Z�⊔IWU&A>�P~�#��dp<�?����7���c��'~���5 ��+$���lx@�M�dm��n<=e�dyX��?{�|Aef ,|n3�<~z�ƃ�uۧ�����P��Y,�ӥQ�*g�#먙R�\���;T��i,��[9Qi歉����c>]9�� ��"�c��P�� �Md?٥��If�ت�u��k��/����F��9�c*9��Ǎ:�ØF���z�n*�@|I�ށ9����N3{'��[�'ͬ�Ҳ4��#}��!�V� Fu��,�,mTIk���v C�7v���B�6k�T9��1�*l� '~��ƞF��lU��'�M ����][ΩũJ_�{�i�I�n��$���L�� j��O�dx�����kza۪��#�E��Cl����x˘�o�����V���ɞ�ljr��)�/,�߬h�L��#��^��L�ф�,íMƁe�̩�NB�L�����iL����q�}��(��q��6IçJ$�W�E$��:������=#����(�K�B����zђ <��K(�N�۫K�w��^O{!����)�H���>x�������lx�?>Պ�+�>�W���,Ly!_�D���Ō�l���Q�!�[ �S����J��1��Ɛ�Y}��b,+�Lo�x�ɓ)����=�y�oh�@�꥟/��I��ѭ=��P�y9��� �ۍYӘ�e+�p�Jnϱ?V\SO%�(�t� ���=?MR�[Ș�����d�/ ��n�l��B�7j� ��!�;ӥ�/�[-���A�>�dN�sLj ��,ɪv��=1c�.SQ�O3�U���ƀ�ܽ�E����������̻��9G�ϷD�7(�}��Ävӌ\�y�_0[w ���<΍>����a_��[0+�L��F.�޺��f�>oN�T����q;���y\��bՃ��y�jH�<|q-eɏ�_?_9+P���Hp$�����[ux�K w�Mw��N�ی'$Y2�=��q���KB��P��~������Yul:�[<����F1�2�O���5=d����]Y�sw:���Ϯ���E��j,_Q��X��z`H1,#II ��d�wr��P˂@�ZJV����y$�\y�{}��^~���[:N����ߌ�U�������O��d�����ؾe��${p>G��3c���Ė�lʌ�� ת��[��`ϱ�-W����dg�I��ig2��� ��}s ��ؤ(%#sS@���~���3�X�nRG�~\jc3�v��ӍL��M[JB�T��s3}��j�Nʖ��W����;7��ç?=X�F=-�=����q�ߚ���#���='�c��7���ڑW�I(O+=:uxq�������������e2�zi+�kuG�R��������0�&e�n���iT^J����~\jy���p'dtG��s����O��3����9* �b#Ɋ�� p������[Bws�T�>d4�ۧs���nv�n���U���_�~,�v����ƜJ1��s�� �QIz��)�(lv8M���U=�;����56��G���s#�K���MP�=��LvyGd��}�VwWBF�'�à �?MH�U�g2�� ����!�p�7Q��j��ڴ����=��j�u��� Jn�A s���uM������e��Ɔ�Ҕ�!)'��8Ϣ�ٔ��ޝ(��Vp���צ֖d=�IC�J�Ǡ{q������kԭ�߸���i��@K����u�|�p=..�*+����x�����z[Aqġ#s2a�Ɗ���RR�)*HRsi�~�a &f��M��P����-K�L@��Z��Xy�'x�{}��Zm+���:�)�) IJ�-i�u���� ���ܒH��'�L(7�y�GӜq���� j��� 6ߌg1�g�o���,kر���tY�?W,���p���e���f�OQS��!K�۟cҒA�|ս�j�>��=⬒��˧L[�� �߿2JaB~R��u�:��Q�] �0H~���]�7��Ƽ�I���(}��cq '�ήET���q�?f�ab���ӥvr� �)o��-Q��_'����ᴎo��K������;��V���o��%���~OK ����*��b�f:���-ťIR��`B�5!RB@���ï�� �u �̯e\�_U�_������� g�ES��3�������QT��a����x����U<~�c?�*�#]�MW,[8O�a�x��]�1bC|踤�P��lw5V%�)�{t�<��d��5���0i�XSU��m:��Z�┵�i�"��1�^B�-��P�hJ��&)O��*�D��c�W��vM��)����}���P��ܗ-q����\mmζZ-l@�}��a��E�6��F�@��&Sg@���ݚ�M����� ȹ 4����#p�\H����dYDo�H���"��\��..R�B�H�z_�/5˘����6��KhJR��P�mƶi�m���3�,#c�co��q�a)*Pt����R�m�k�7x�D�E�\Y�閣_X�<���~�)���c[[�BP����6�Yq���S��0����%_����;��Àv�~�| VS؇ ��'O0��F0��\���U�-�d@�����7�SJ*z��3n��y��P����O���������m�~�P�3|Y��ʉr#�C�<�G~�.,! ���bqx���h~0=��!ǫ�jy����l�O,�[B��~��|9��ٱ����Xly�#�i�B��g%�S��������tˋ���e���ې��\[d�t)��.+u�|1 ������#�~Oj����hS�%��i.�~X���I�H�m��0n���c�1uE�q��cF�RF�o���7� �O�ꮧ� ���ۛ{��ʛi5�rw?׌#Qn�TW��~?y$��m\�\o����%W� ?=>S�N@�� �Ʈ���R����N�)�r"C�:��:����� �����#��qb��Y�. �6[��2K����2u�Ǧ�HYR��Q�MV��� �G�$��Q+.>�����nNH��q�^��� ����q��mM��V��D�+�-�#*�U�̒ ���p욳��u:�������IB���m���PV@O���r[b= �� ��1U�E��_Nm�yKbN�O���U�}�the�`�|6֮P>�\2�P�V���I�D�i�P�O;�9�r�mAHG�W�S]��J*�_�G��+kP�2����Ka�Z���H�'K�x�W�MZ%�O�YD�Rc+o��?�q��Ghm��d�S�oh�\�D�|:W������UA�Qc yT�q������~^�H��/��#p�CZ���T�I�1�ӏT����4��"�ČZ�����}��`w�#�*,ʹ�� ��0�i��課�Om�*�da��^gJ݅{���l�e9uF#T�ֲ��̲�ٞC"�q���ߍ ոޑ�o#�XZTp����@ o�8��(jd��xw�]�,f���`~�|,s��^����f�1���t��|��m�򸄭/ctr��5s��7�9Q�4�H1꠲BB@l9@���C�����+�wp�xu�£Yc�9��?`@#�o�mH�s2��)�=��2�.�l����jg�9$�Y�S�%*L������R�Y������7Z���,*=�䷘$�������arm�o�ϰ���UW.|�r�uf����IGw�t����Zwo��~5 ��YյhO+=8fF�)�W�7�L9lM�̘·Y���֘YLf�큹�pRF���99.A �"wz��=E\Z���'a� 2��Ǚ�#;�'}�G���*��l��^"q��+2FQ� hj��kŦ��${���ޮ-�T�٭cf�|�3#~�RJ����t��$b�(R��(����r���dx� >U b�&9,>���%E\� Ά�e�$��'�q't��*�א���ެ�b��-|d���SB�O�O��$�R+�H�)�܎�K��1m`;�J�2�Y~9��O�g8=vqD`K[�F)k�[���1m޼c��n���]s�k�z$@��)!I �x՝"v��9=�ZA=`Ɠi �:�E��)`7��vI��}d�YI�_ �o�:ob���o ���3Q��&D&�2=�� �Ά��;>�h����y.*ⅥS������Ӭ�+q&����j|UƧ����}���J0��WW< ۋS�)jQR�j���Ư��rN)�Gű�4Ѷ(�S)Ǣ�8��i��W52���No˓� ۍ%�5brOn�L�;�n��\G����=�^U�dI���8$�&���h��'���+�(������cȁ߫k�l��S^���cƗjԌE�ꭔ��gF���Ȓ��@���}O���*;e�v�WV���YJ\�]X'5��ղ�k�F��b 6R�o՜m��i N�i����>J����?��lPm�U��}>_Z&�KK��q�r��I�D�Չ~�q�3fL�:S�e>���E���-G���{L�6p�e,8��������QI��h��a�Xa��U�A'���ʂ���s�+טIjP�-��y�8ۈZ?J$��W�P� ��R�s�]��|�l(�ԓ��sƊi��o(��S0��Y� 8�T97.�����WiL��c�~�dxc�E|�2!�X�K�Ƙਫ਼�$((�6�~|d9u+�qd�^3�89��Y�6L�.I�����?���iI�q���9�)O/뚅����O���X��X�V��ZF[�یgQ�L��K1���RҖr@v�#��X�l��F���Нy�S�8�7�kF!A��sM���^rkp�jP�DyS$N���q��nxҍ!U�f�!eh�i�2�m���`�Y�I�9r�6� �TF���C}/�y�^���Η���5d�'��9A-��J��>{�_l+�`��A���[�'��յ�ϛ#w:݅�%��X�}�&�PSt�Q�"�-��\縵�/����$Ɨh�Xb�*�y��BS����;W�ջ_mc�����vt?2}1�;qS�d�d~u:2k5�2�R�~�z+|HE!)�Ǟl��7`��0�<�,�2*���Hl-��x�^����'_TV�gZA�'j� ^�2Ϊ��N7t�����?w�� �x1��f��Iz�C-Ȗ��K�^q�;���-W�DvT�7��8�Z�������� hK�(P:��Q- �8�n�Z���܃e貾�<�1�YT<�,�����"�6{/ �?�͟��|1�:�#g��W�>$����d��J��d�B��=��jf[��%rE^��il:��B���x���Sּ�1հ��,�=��*�7 fcG��#q� �eh?��2�7�����,�!7x��6�n�LC�4x��},Geǝ�tC.��vS �F�43��zz\��;QYC,6����~;RYS/6���|2���5���v��T��i����������mlv��������&� �nRh^ejR�LG�f���? �ۉҬܦƩ��|��Ȱ����>3����!v��i�ʯ�>�v��オ�X3e���_1z�Kȗ\<������!�8���V��]��?b�k41�Re��T�q��mz��TiOʦ�Z��Xq���L������q"+���2ۨ��8}�&N7XU7Ap�d�X��~�׿��&4e�o�F��� �H����O���č�c�� 懴�6���͉��+)��v;j��ݷ�� �UV�� i��� j���Y9GdÒJ1��詞�����V?h��l����l�cGs�ځ�������y�Ac�����\V3�? �� ܙg�>qH�S,�E�W�[�㺨�uch�⍸�O�}���a��>�q�6�n6����N6�q������N ! 1AQaq�0@����"2BRb�#Pr���3C`��Scst���$4D���%Td�� ?���N����a��3��m���C���w��������xA�m�q�m���m������$����4n淿t'��C"w��zU=D�\R+w�p+Y�T�&�պ@��ƃ��3ޯ?�Aﶂ��aŘ���@-�����Q�=���9D��ռ�ѻ@��M�V��P��܅�G5�f�Y<�u=,EC)�<�Fy'�"�&�չ�X~f��l�KԆV��?�� �W�N����=(� �;���{�r����ٌ�Y���h{�١������jW����P���Tc�����X�K�r��}���w�R��%��?���E��m�� �Y�q|����\lEE4���r���}�lsI�Y������f�$�=�d�yO����p�����yBj8jU�o�/�S��?�U��*������ˍ�0������u�q�m [�?f����a�� )Q�>����6#������� ?����0UQ����,IX���(6ڵ[�DI�MNލ�c&���υ�j\��X�R|,4��� j������T�hA�e��^���d���b<����n�� �즇�=!���3�^�`j�h�ȓr��jẕ�c�,ٞX����-����a�ﶔ���#�$��]w�O��Ӫ�1y%��L�Y<�wg#�ǝ�̗`�x�xa�t�w��»1���o7o5��>�m뭛C���Uƃߜ}�C���y1Xνm�F8�jI���]����H���ۺиE@I�i;r�8ӭ����V�F�Շ| ��&?�3|x�B�MuS�Ge�=Ӕ�#BE5G�����Y!z��_e��q�р/W>|-�Ci߇�t�1ޯќd�R3�u��g�=0 5��[?�#͏��q�cf���H��{ ?u�=?�?ǯ���}Z��z���hmΔ�BFTW�����<�q�(v� ��!��z���iW]*�J�V�z��gX֧A�q�&��/w���u�gYӘa���; �i=����g:��?2�dž6�ى�k�4�>�Pxs����}������G�9��3 ���)gG�R<>r h�$��'nc�h�P��Bj��J�ҧH� -��N1���N��?��~��}-q!=��_2hc�M��l�vY%UE�@|�v����M2�.Y[|y�"Eï��K�ZF,�ɯ?,q�?v�M 80jx�"�;�9vk�����+ ֧�� �ȺU��?�%�vcV��mA�6��Qg^M����A}�3�nl� QRN�l8�kkn�'�����(��M�7m9و�q���%ޟ���*h$Zk"��$�9��: �?U8�Sl��,,|ɒ��xH(ѷ����Gn�/Q�4�P��G�%��Ա8�N��!� �&�7�;���eKM7�4��9R/%����l�c>�x;������>��C�:�����t��h?aKX�bhe�ᜋ^�$�Iհ �hr7%F$�E��Fd���t��5���+�(M6�t����Ü�UU|zW�=a�Ts�Tg������dqP�Q����b'�m���1{|Y����X�N��b �P~��F^F:����k6�"�j!�� �I�r�`��1&�-$�Bevk:y���#yw��I0��x��=D�4��tU���P�ZH��ڠ底taP��6����b>�xa����Q�#� WeF��ŮNj�p�J* mQ�N����*I�-*�ȩ�F�g�3 �5��V�ʊ�ɮ�a��5F���O@{���NX��?����H�]3��1�Ri_u��������ѕ�� ����0��� F��~��:60�p�͈�S��qX#a�5>���`�o&+�<2�D����: �������ڝ�$�nP���*)�N�|y�Ej�F�5ټ�e���ihy�Z �>���k�bH�a�v��h�-#���!�Po=@k̆IEN��@��}Ll?j�O������߭�ʞ���Q|A07x���wt!xf���I2?Z��<ץ�T���cU�j��]��陎Ltl �}5�ϓ��$�,��O�mˊ�;�@O��jE��j(�ا,��LX���LO���Ц�90�O �.����a��nA���7������j4 ��W��_ٓ���zW�jcB������y՗+EM�)d���N�g6�y1_x��p�$Lv:��9�"z��p���ʙ$��^��JԼ*�ϭ����o���=x�Lj�6�J��u82�A�H�3$�ٕ@�=Vv�]�'�qEz�;I˼��)��=��ɯ���x �/�W(V���p�����$ �m�������u�����񶤑Oqˎ�T����r��㠚x�sr�GC��byp�G��1ߠ�w e�8�$⿄����/�M{*}��W�]˷.�CK\�ުx���/$�WPw���r� |i���&�}�{�X� �>��$-��l���?-z���g����lΆ���(F���h�vS*���b���߲ڡn,|)mrH[���a�3�ר�[1��3o_�U�3�TC�$��(�=�)0�kgP���� ��u�^=��4 �WYCҸ:��vQ�ר�X�à��tk�m,�t*��^�,�}D*� �"(�I��9R����>`�`��[~Q]�#af��i6l��8���6�:,s�s�N6�j"�A4���IuQ��6E,�GnH��zS�HO�uk�5$�I�4��ؤ�Q9�@��C����wp�BGv[]�u�Ov���0I4���\��y�����Q�Ѹ��~>Z��8�T��a��q�ޣ;z��a���/��S��I:�ܫ_�|������>=Z����8:�S��U�I�J��"IY���8%b8���H��:�QO�6�;7�I�S��J��ҌAά3��>c���E+&jf$eC+�z�;��V����� �r���ʺ������my�e���aQ�f&��6�ND��.:��NT�vm�<- u���ǝ\MvZY�N�NT��-A�>jr!S��n�O 1�3�Ns�%�3D@���`������ܟ 1�^c<���� �a�ɽ�̲�Xë#�w�|y�cW�=�9I*H8�p�^(4���՗�k��arOcW�tO�\�ƍR��8����'�K���I�Q�����?5�>[�}��yU�ײ -h��=��% q�ThG�2�)���"ו3]�!kB��*p�FDl�A���,�eEi�H�f�Ps�����5�H:�Փ~�H�0Dت�D�I����h�F3�������c��2���E��9�H��5�zԑ�ʚ�i�X�=:m�xg�hd(�v����׊�9iS��O��d@0ڽ���:�p�5�h-��t�&���X�q�ӕ,��ie�|���7A�2���O%P��E��htj��Y1��w�Ѓ!����  ���� ࢽ��My�7�\�a�@�ţ�J �4�Ȼ�F�@o�̒?4�wx��)��]�P��~�����u�����5�����7X ��9��^ܩ�U;Iꭆ 5 �������eK2�7(�{|��Y׎ �V��\"���Z�1� Z�����}��(�Ǝ"�1S���_�vE30>���p;� ΝD��%x�W�?W?v����o�^V�i�d��r[��/&>�~`�9Wh��y�;���R��� ;;ɮT��?����r$�g1�K����A��C��c��K��l:�'��3 c�ﳯ*"t8�~l��)���m��+U,z��`(�>yJ�?����h>��]��v��ЍG*�{`��;y]��I�T� ;c��NU�fo¾h���/$���|NS���1�S�"�H��V���T���4��uhǜ�]�v;���5�͠x��'C\�SBpl���h}�N����� A�Bx���%��ޭ�l��/����T��w�ʽ]D�=����K���ž�r㻠l4�S�O?=�k �M:� ��c�C�a�#ha���)�ѐxc�s���gP�iG��{+���x���Q���I= �� z��ԫ+ �8"�k�ñ�j=|����c ��y��CF��/��*9ж�h{ �?4�o� ��k�m�Q�N�x��;�Y��4膚�a�w?�6�>e]�����Q�r�:����g�,i"�����ԩA�*M�<�G��b�if��l^M��5� �Ҩ�{����6J��ZJ�����P�*�����Y���ݛu�_4�9�I8�7���������,^ToR���m4�H��?�N�S�ѕw��/S��甍�@�9H�S�T��t�ƻ���ʒU��*{Xs�@����f�����֒Li�K{H�w^���������Ϥm�tq���s� ���ք��f:��o~s��g�r��ט� �S�ѱC�e]�x���a��) ���(b-$(�j>�7q�B?ӕ�F��hV25r[7 Y� }L�R��}����*sg+��x�r�2�U=�*'WS��ZDW]�WǞ�<��叓���{�$�9Ou4��y�90-�1�'*D`�c�^o?(�9��u���ݐ��'PI&� f�Jݮ�������:wS����jfP1F:X �H�9dԯ���˝[�_54 �}*;@�ܨ�� ð�yn�T���?�ןd�#���4rG�ͨ��H�1�|-#���Mr�S3��G�3�����)�.᧏3v�z֑��r����$G"�`j �1t��x0<Ɔ�Wh6�y�6��,œ�Ga��gA����y��b��)��h�D��ß�_�m��ü �gG;��e�v��ݝ�nQ� ��C����-�*��o���y�a��M��I�>�<���]obD��"�:���G�A��-\%LT�8���c�)��+y76���o�Q�#*{�(F�⽕�y����=���rW�\p���۩�c���A���^e6��K������ʐ�cVf5$�'->���ՉN"���F�"�UQ@�f��Gb~��#�&�M=��8�ט�JNu9��D��[̤�s�o�~������ G��9T�tW^g5y$b��Y'��س�Ǵ�=��U-2 #�MC�t(�i� �lj�@Q 5�̣i�*�O����s�x�K�f��}\��M{E�V�{�υ��Ƈ�����);�H����I��fe�Lȣr�2��>��W�I�Ȃ6������i��k�� �5�YOxȺ����>��Y�f5'��|��H+��98pj�n�.O�y�������jY��~��i�w'������l�;�s�2��Y��:'lg�ꥴ)o#'Sa�a�K��Z� �m��}�`169�n���"���x��I ��*+� }F<��cГ���F�P�������ֹ*�PqX�x۩��,� ��N�� �4<-����%����:��7����W���u�`����� $�?�I��&����o��o��`v�>��P��"��l���4��5'�Z�gE���8���?��[�X�7(��.Q�-��*���ތL@̲����v��.5���[��=�t\+�CNܛ��,g�SQnH����}*F�G16���&:�t��4ُ"A��̣��$�b �|����#rs��a�����T�� ]�<�j��BS�('$�ɻ� �wP;�/�n��?�ݜ��x�F��yUn�~mL*-�������Xf�wd^�a�}��f�,=t�׵i�.2/wpN�Ep8�OР���•��R�FJ� 55TZ��T �ɭ�<��]��/�0�r�@�f��V��V����Nz�G��^���7hZi����k��3�,kN�e|�vg�1{9]_i��X5y7� 8e]�U����'�-2,���e"����]ot�I��Y_��n�(JҼ��1�O ]bXc���Nu�No��pS���Q_���_�?i�~�x h5d'�(qw52] ��'ޤ�q��o1�R!���`ywy�A4u���h<קy���\[~�4�\ X�Wt/� 6�����n�F�a8��f���z �3$�t(���q��q�x��^�XWeN'p<-v�!�{�(>ӽDP7��ո0�y)�e$ٕv�Ih'Q�EA�m*�H��RI��=:��� ���4牢) �%_iN�ݧ�l]� �Nt���G��H�L��� ɱ�g<���1V�,�J~�ٹ�"K��Q�� 9�HS�9�?@��k����r�;we݁�]I�!{ �@�G�[�"��`���J:�n]�{�cA�E����V��ʆ���#��U9�6����j�#Y�m\��q�e4h�B�7��C�������d<�?J����1g:ٳ���=Y���D�p�ц� ׈ǔ��1�]26؜oS�'��9�V�FVu�P�h�9�xc�oq�X��p�o�5��Ա5$�9W�V(�[Ak�aY錎qf;�'�[�|���b�6�Ck��)��#a#a˙��8���=äh�4��2��C��4tm^ �n'c���]GQ$[Wҿ��i���vN�{Fu ��1�gx��1┷���N�m��{j-,��x�� Ūm�ЧS�[�s���Gna���䑴�� x�p 8<������97�Q���ϴ�v�aϚG��Rt�Һ׈�f^\r��WH�JU�7Z���y)�vg=����n��4�_)y��D'y�6�]�c�5̪�\� �PF�k����&�c;��cq�$~T�7j ���nç]�<�g ":�to�t}�159�<�/�8������m�b�K#g'I'.W�����6��I/��>v��\�MN��g���m�A�yQL�4u�Lj�j9��#44�t��l^�}L����n��R��!��t��±]��r��h6ٍ>�yҏ�N��fU�� ���� Fm@�8}�/u��jb9������he:A�y�ծw��GpΧh�5����l}�3p468��)U��d��c����;Us/�֔�YX�1�O2��uq�s��`hwg�r~�{ R��mhN��؎*q 42�*th��>�#���E����#��Hv�O����q�}�����6�e��\�,Wk�#���X��b>��p}�դ��3���T5��†��6��[��@�P�y*n��|'f�֧>�lư΂�̺����SU�'*�q�p�_S�����M�� '��c�6�����m�� ySʨ;M��r���Ƌ�m�Kxo,���Gm�P��A�G�:��i��w�9�}M(�^�V��$ǒ�ѽ�9���|���� �a����J�SQ�a���r�B;����}���ٻ֢�2�%U���c�#�g���N�a�ݕ�'�v�[�OY'��3L�3�;,p�]@�S��{ls��X�'���c�jw�k'a�.��}�}&�� �dP�*�bK=ɍ!����;3n�gΊU�ߴmt�'*{,=SzfD� A��ko~�G�aoq�_mi}#�m�������P�Xhύ����mxǍ�΂���巿zf��Q���c���|kc�����?���W��Y�$���_Lv����l߶��c���`?����l�j�ݲˏ!V��6����U�Ђ(A���4y)H���p�Z_�x��>���e��R��$�/�`^'3qˏ�-&Q�=?��CFVR �D�fV�9��{�8g�������n�h�(P"��6�[�D���< E�����~0<@�`�G�6����Hг�cc�� �c�K.5��D��d�B���`?�XQ��2��ٿyqo&+�1^� DW�0�ꊩ���G�#��Q�nL3��c���������/��x ��1�1[y�x�პCW��C�c�UĨ80�m�e�4.{�m��u���I=��f�����0QRls9���f���������9���~f�����Ǩ��a�"@�8���ȁ�Q����#c�ic������G��$���G���r/$W�(��W���V�"��m�7�[m�A�m����bo��D� j����۳� l���^�k�h׽����� ��#� iXn�v��eT�k�a�^Y�4�BN��ĕ��0 !01@Q"2AaPq3BR������?���@4�Q�����T3,���㺠�W�[=JK�Ϟ���2�r^7��vc�:�9 �E�ߴ�w�S#d���Ix��u��:��Hp��9E!�� V 2;73|F��9Y���*ʬ�F��D����u&���y؟��^EA��A��(ɩ���^��GV:ݜDy�`��Jr29ܾ�㝉��[���E;Fzx��YG��U�e�Y�C���� ����v-tx����I�sם�Ę�q��Eb�+P\ :>�i�C'�;�����k|z�رn�y]�#ǿb��Q��������w�����(�r|ӹs��[�D��2v-%��@;�8<a���[\o[ϧw��I!��*0�krs)�[�J9^��ʜ��p1)� "��/_>��o��<1����A�E�y^�C��`�x1'ܣn�p��s`l���fQ��):�l����b>�Me�jH^?�kl3(�z:���1ŠK&?Q�~�{�ٺ�h�y���/�[��V�|6��}�KbX����mn[-��7�5q�94�������dm���c^���h� X��5��<�eޘ>G���-�}�دB�ޟ� ��|�rt�M��V+�]�c?�-#ڛ��^ǂ}���Lkr���O��u�>�-D�ry� D?:ޞ�U��ǜ�7�V��?瓮�"�#���r��չģVR;�n���/_� ؉v�ݶe5d�b9��/O��009�G���5n�W����JpA�*�r9�>�1��.[t���s�F���nQ� V 77R�]�ɫ8����_0<՜�IF�u(v��4��F�k�3��E)��N:��yڮe��P�`�1}�$WS��J�SQ�N�j�ٺ��޵�#l���ј(�5=��5�lǏmoW�v-�1����v,W�mn��߀$x�<����v�j(����c]��@#��1������Ǔ���o'��u+����;G�#�޸��v-lη��/(`i⣍Pm^���ԯ̾9Z��F��������n��1��� ��]�[��)�'������:�֪�W��FC����� �B9،!?���]��V��A�Վ�M��b�w��G F>_DȬ0¤�#�QR�[V��kz���m�w�"��9ZG�7'[��=�Q����j8R?�zf�\a�=��O�U����*oB�A�|G���2�54 �p��.w7� �� ��&������ξxGHp� B%��$g�����t�Џ򤵍z���HN�u�Я�-�'4��0��;_��3 !01"@AQa2Pq#3BR������?��ʩca��en��^��8���<�u#��m*08r��y�N"�<�Ѳ0��@\�p��� �����Kv�D��J8�Fҽ� �f�Y��-m�ybX�NP����}�!*8t(�OqѢ��Q�wW�K��ZD��Δ^e��!� ��B�K��p~�����e*l}z#9ң�k���q#�Ft�o��S�R����-�w�!�S���Ӥß|M�l޶V��!eˈ�8Y���c�ЮM2��tk���� ������J�fS����Ö*i/2�����n]�k�\���|4yX�8��U�P.���Ы[���l��@"�t�<������5�lF���vU�����W��W��;�b�cД^6[#7@vU�xgZv��F�6��Q,K�v��� �+Ъ��n��Ǣ��Ft���8��0��c�@�!�Zq s�v�t�;#](B��-�nῃ~���3g������5�J�%���O������n�kB�ĺ�.r��+���#�N$?�q�/�s�6��p��a����a��J/��M�8��6�ܰ"�*������ɗud"\w���aT(����[��F��U՛����RT�b���n�*��6���O��SJ�.�ij<�v�MT��R\c��5l�sZB>F��<7�;EA��{��E���Ö��1U/�#��d1�a�n.1ě����0�ʾR�h��|�R��Ao�3�m3 ��%�� ���28Q� ��y��φ���H�To�7�lW>����#i`�q���c����a��� �m,B�-j����݋�'mR1Ήt�>��V��p���s�0IbI�C.���1R�ea�����]H�6����������4B>��o��](��$B���m�����a�!=��?�B� K�Ǿ+�Ծ"�n���K��*��+��[T#�{E�J�S����Q�����s�5�:�U�\wĐ�f�3����܆&�)����I���Ԇw��E T�lrTf6Q|R�h:��[K�� �z��c֧�G�C��%\��_�a�84��HcO�bi��ؖV��7H �)*ģK~Xhչ0��4?�0��� �E<���}3���#���u�?�� ��|g�S�6ꊤ�|�I#Hڛ� �ա��w�X��9��7���Ŀ%�SL��y6č��|�F�a 8���b��$�sק�h���b9RAu7�˨p�Č�_\*w��묦��F ����4D~�f����|(�"m���NK��i�S�>�$d7SlA��/�²����SL��|6N�}���S�˯���g��]6��; �#�.��<���q'Q�1|KQ$�����񛩶"�$r�b:���N8�w@��8$�� �AjfG|~�9F ���Y��ʺ��Bwؒ������M:I岎�G��`s�YV5����6��A �b:�W���G�q%l�����F��H���7�������Fsv7��k�� 403WebShell
403Webshell
Server IP : 172.67.215.126  /  Your IP : 172.69.214.109
Web Server : Apache/2.4.52 (Ubuntu)
System : Linux ip-172-31-19-221 6.8.0-1029-aws #31~22.04.1-Ubuntu SMP Thu Apr 24 21:16:18 UTC 2025 x86_64
User : www-data ( 33)
PHP Version : 8.1.28
Disable Function : NONE
MySQL : OFF  |  cURL : ON  |  WGET : ON  |  Perl : ON  |  Python : OFF  |  Sudo : ON  |  Pkexec : ON
Directory :  /efsdata/scitechnol.com/httpdocs/submission/uploads/

Upload File :
current_dir [ Writeable ] document_root [ Writeable ]

 

Command :


[ Back ]     

Current File : /efsdata/scitechnol.com/httpdocs/submission/uploads/1255-1-Manuscript-2013-10-16.doc
��ࡱ�>��	����������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������bjbj�W�WA��=�=Re,�������xx������������8��t��4�22HHH#>a}�3�3�3�3�3�3�3$�6�69B#4E��##��#4��HH�h4i.i.i.�&�H�H�3i.��3i.i.:�2,-3H�����-������*��2
�3~40�4�2.x9U,x9-3-3x9�A3�i.���#4#4i.����4������������������������������������������������������������������������x9���������x�:Title: Comparison of different methods for exogenous DNA uptake by bovine spermatozoa 
Authors: Renata Sim�es1,2; Weber Beringui Feitosa1; Marcella Pecora Milazzotto1,2; Alessandra Corallo Nicacio1; Flavia Regina Oliveira de Barros1, Jos� S�rgio de Arruda Gon�alves1; Mariana Groke Marques1; Jos� Ant�nio Visintin1; Mayra Elena Ortiz D��vila Assump��o1
Authors� affilitations
1 Centro de Ci�ncias Naturais e Humanas (CCNH), Federal University of ABC. Rua Santa Ad�lia, 166. Bairro Bangu. Santo Andr� - SP - Brazil. CEP 09210-170. 
2 College of Veterinary Medicine, University of S�o Paulo, Department of Animal Reproduction. Av. Prof. Dr. Orlando Marques de Paiva, 87 � Cidade Universit�ria, S�o Paulo/SP � Brasil CEP: 05508-270
Corresponding author�s postal and email: 
Mayra Elena Ortiz D��vila Assump��o 
Faculdade de Medicina Veterin�ria e Zootecnia da Universidade de S�o Paulo
Departamento de Reprodu��o Animal
Av. Prof. Dr. Orlando Marques de Paiva, 87 � Cidade Universit�ria, S�o Paulo/SP � Brasil CEP: 05508-270
Email: meoaa@usp.br
Institution where the work was carried out:�College of Veterinary Medicine, University of S�o Paulo, Department of Animal Reproduction.
�short title:  bovine sperm as a vector & animal transgenesis





ABSTRACT
Although genetic manipulation of farm animals is of great interest for animal production and pharmaceutical industry, its efficiency remains far from the satisfactory. Pronuclear injection, which is the most used technique for such modification, mainly in mice, remains limited for this specie. Some alternatives have been developed such as sperm mediated gene transfer, in which the spermatozoa are used as vectors for DNA delivery during in vitro fertilization. Mature sperm cells are able to spontaneously bind exogenous DNA molecules which may be internalized into sperm nuclei. Given the potential of sperm mediated gene transfer for livestock animals transgenesis, the aim of this study was to evaluate four methods of DNA uptake for sperm mediated gene transfer in bovine: incubation with DNA, plasma membrane alteration induced by calcium ionophore followed by incubation with DNA, electroporation and lipofection. Spermatozoa not exposed to exogenous DNA were used as control group. Cleavage, blastocyst and hatching rates were recorded at 72 hours post insemination (hpi), Days 9 and 12 of embryo culture, respectively. Exogenous DNA positive embryos were evaluated by PCR. No effect of treatment was observed on cleavage, blastocyst and hatching rates. In addition, percentage of DNA positive blastocysts did not differ among experimental groups. In spite of the low number of positive embryos, our results show that all treatments presented similar efficiencies for DNA delivery during in vitro fertilization. In conclusion, although the development rates were similar and constant in all groups, other factors such as exogenous DNA sequence, size and concentration should be considered to improve sperm mediated gene transfer.

Key-words: bovine, transgenesis, SMGT, incubation, calcium ionophore, electroporation, lipofection, IVP.
INTRODUCTION

Introducing exogenous DNA in farm animal cells, particularly in the germ line, is of great value for agricultural production, development of xenotransplant tecniques, generation of bioreactors for the pharmaceutical industry [1, 2]. However, the methods generally used for this purpose present low efficiency, are laborious and expensive [3, 4] in these species.
The use of spermatozoa as a vector of exogenous DNA was described as a simple and cheaper alternative for livestock transgenic technology [5]. The foreign DNA can be either integrated into the spermatozoa chromosomal DNA or simply carried to the egg by the spermatozoa and to be integrated later in the zygote's genome [6]. In 1989, Arezzo and Lavitrano et al. [7] described that sperm cells spontaneously bind to exogenous DNA molecules and transfer them to the oocyte during in vitro fertilization (IVF), generating transgenic offspring. However, the efficiency of this technique remains low and inconsistent. Efficiencies of <1% required microinjection and transfer of thousands of embryos to produce a few transgenic offspring [4, 8].
Several protocols have been used to improve the production of transgenic embryos using sperm mediated gene transfer (SMGT). In 1991, Gagn� et al. [9] described exogenous DNA uptake by bovine spermatozoa after electroporation. Electroporation is a well established in vitro technique and is used to introduce DNA or other molecules into cells. A high electric field pulses cause a transient increase in the permeability of cell membranes, leading to pore formation and augmented permeability to ions, molecules and even macromolecules as exogenous DNA [10].
Rottmann et al. [11] obtained genetic modified mice offspring after treatment of sperm cells with artificial vesicles called liposomes. These cationic lipids interact with the negatively charged nucleic acid molecules, protecting the DNA from proteases and DNases. The positive outer surface of the liposome can then associate with the negative charged cell membrane allowing the internalization and further delivery of exogenous materials in to the cells, including transgenes [12-14]. 
Plasma membrane modification induced by Calcium ionophore (CaI) aiming the production of genetic modified animals is rarely reported in literature [15]. This is perhaps because CaI leads to some morphological and functional changes in sperm plasma membrane (capacitation and induced acrosome reaction) that could result in decreased oocyte fertilization [16]. However, Perry et al. [17] demonstrated that sperm with membrane disruption are able to support full embryo development. In addition, Pereira et al. [18] reported that bull and goat sperm cells treated with CaI showed a significantly improved percentage of live spermatozoa with true acrosome reaction. This information can be of great value for sperm exogenous DNA uptake since during the acrosome reaction several changes occur in membrane fluidity which may facilitate the entry of the transgene.  
In order to improve the efficiency of SMGT in bovine, the objective of the present study was to evaluate four methods of sperm DNA uptake: electroporation, lipofection, plasma membrane modification induced by CaI followed by incubation with DNA and incubation; and their effects on embryo development in vitro.

MATERIAL AND METHODS

 A total of 1513 oocytes were subjected to in vitro production (IVP) of embryos (mean of 302.6 oocytes per group distributed in 10 replicates). Unless otherwise indicated, all chemicals were obtained from Sigma Chemical (St. Louis, MO, USA). Tissue culture media (TCM), Hepes and Bicarbonate and fetal calf serum (FCS) were obtained from Gibco (Grand Island, NY, USA). Recombinant plasmid DNA (pEYFP-NUC) was obtained from Clonetech, BD Biosciences (USA). All experiments were performed using 500ng of recombinant plasmid DNA as exogenous DNA, which was 4.8 kb in size and linearized with Stu I at 37oC for 2 h. These experiments were approved by bioethics committee of the School of Veterinary Medicine and Animal Sciences, University of S�o Paulo (protocol number 400/2003).

Sperm preparation and exogenous DNA uptake
Frozen-thawed semen was obtained from one bull. The thawed semen (37oC in water bath / 30 sec) was centrifuged at 600 x g for 30 min in Percoll gradient (45% and 90%) to purify viable sperm cells. The pellet was washed in Sperm-TALP [19] followed by centrifugation at 200 x g for 5 min. The pellet was resuspended in Sperm-TALP to a 5x106 spermatozoa/mL final concentration for each of the experimental groups: Control (spermatozoa not exposed to exogenous DNA), electroporation (E) lipofection (L), plasma membrane modification induced by calcium ionophore followed by incubation with DNA (CaI), and incubation (I).

Electroporation
A concentration of 5x106 spermatozoa/mL was added to 1mL of TCM199 and 500ng of exogenous DNA. Electroporation was accomplished with one pulse of 12msec, 500 V and 25 �F (Cellject Pro Hybaid, Franklin, MA, USA).

Lipofection
Effectene� (Qiagen, Canada) reagent was used for lipofection following manufacture recommendation. Briefly, 10uL of liposome were incubated with 500ng of exogenous DNA for 10 min at room temperature. Sperm cells were added to the liposome-exogenous DNA mix and incubated for 2 hours at 39�C, 5% CO2 in air (v/v) and high humidity.

Calcium ionophore (CaI)
Sperm cells were incubated in 1mL of in vitro fertilization (IVF) medium supplemented with 250nM of CaI (A23187) for 5 min, centrifuged at 200g for 5 min, resuspended in Sperm-TALP and centrifuged again. The pellet was resuspended in 1mL of IVF medium (IVF-Talp - [19]) without heparin containing 500ng of exogenous DNA at 39�C, 5% CO2 in air (v/v) and high humidity for 1h.

Incubation
Sperm cells were added to 1mL of IVF medium without heparin and 500ng of exogenous DNA for 1h at 39�C, 5% CO2 in air (v/v) and high humidity.

Oocyte in vitro maturation
Embryos were produced according to Yamada et al. [20]. Briefly, cumulus oocyte complexes (COCs) were obtained by aspirating follicles from abattoir ovaries. Groups of 15-30 COCs with homogeneous ooplasm surrounded with multilayer compacted cumulus cells were placed in 90��L maturation medium (TCM 199 Bicarbonate supplemented with 10% (v/v) FCS, 22��g/ml pyruvate, 50��g/ml gentamycin, 0.5��g/ml FSH (Folltropin-V; Bioniche Life Science Inc., Ontario, Canada), 50��g/ml hCG (Chorulon,  MSD-Animal Health, The Netherlands) and 1��g/ml 17 beta estradiol), covered with mineral oil and cultured for 24�h at 38.5��C under an atmosphere of 5% CO2 in air and high humidity.

In vitro fertilization
For in vitro fertilization, COCs were washed three times in fertilization medium before being transferred in groups of 15-30 oocytes, into plates containing 90�L of fertilization medium IVF-Talp: modified tyrode stock solution supplemented with PHE (0.5mM penicillamine; 0.25mM hypotaurine and 25�M epinephrine in 0.9% (w/v) NaCl), 50�g/mL gentamycin and 0.3% essential fatty acid free bovine serum albumin, under mineral oil. After treatments, spermatozoa from experimental and control groups were washed by centrifugation in Sperm-TALP and in IVF medium at 200 x g for 5 min. Oocytes were inseminated with 1X106 spermatozoa/mL (for all experimental and control groups). IVF was carried out at 38.5��C in a 5% CO2 atmosphere in air and high humidity.

In vitro embryo culture (IVC)
At approximately 18 hpi, presumptive zygotes were partially denuded by gentle pipetting and washed three times in SOFaa medium [21], supplemented with 5% (v/v) FCS, MEM nonessential amino acids and MEM essential amino acids solution before being transferred to 90�L drop of culture medium (SOFaa) in the same plate used for IVM (co-culture system) under mineral oil, at 38.5��C in a 5% CO2 atmosphere and high humidity. IVC was carried out for 12 days being IVF considered as Day 0. Cleavage rate was recorded at 72 hpi and blastocyst and hatching rates were respectively recorded at Days 9 and 12.
Blastocysts were collected at day 12 of IVC and washed individually in PBS drops of 20�L and frozen in microcentrifuge tubes. After extraction of DNA, polymerase chain reaction (PCR) was performed to assess the presence of exogenous DNA in Day 12 embryos. The experiment was repeated ten times including all treatments.
PCR
A 272bp plasmid DNA fragment was amplified with specific primers (IDT, IA, USA): 5�- GGCAAGCTGACCCTGAAGTT � 3� (forward) and 5�- AAGTCGATGCCCTTCAGCTC � 3� (reverse). Each amplification mixture (30�L) was prepared into the tubes containing the frozen-thawed embryos, 1.25mM of each dNTP (dATP, dCTP, dTTP and dGTP - Invitrogen, USA), 2UI of Taq DNA polimerase (Invitrogen, USA), 20pmol of each specific primer, 3�L of 10x buffer (200mM Tris-HCl � pH 8.0; 500mM KCl � Invitrogen, USA) and 1.5mM of MgCl2 (Invitrogen, USA). PCR amplification was performed with a DNA thermal cycler (PTC-100 MJ Research) for 35 cycles of 95oC for 1min, annealing at 60oC for 1min and extension at 72oC for 1min. PCR mixtures without sample were used as PCR negative control.
PCR from control group embryos was carried out as negative control of treatments. The absence of false positive embryos was checked by PCR reactions of culture medium (SOFaa) and washing medium (PBS). Plasmidial DNA was amplified as PCR positive control. PCR products were submitted to 1% agarose gel electrophoresis stained with ethidium bromide.

DNA sequencing
The amplified products (272bp) were sliced from the gel and purified with Rapid Gel Extration System (Marligen Biosciences Inc., USA). The purified product (5�L) was added to 5pMol of forward or reverse specific primer and 2�L of distilled water and sequenced by BigDye2 dideoxyterminators system (Applied Biosystems, CA, USA). At least one sample from each group was sequenced.

Statistical Analysis
All data were evaluated using SAS System for Windows (SAS Institute Inc., Cary, NC, USA). Residue normality (Gaussian distribution) and variance homogeneity of each variable were evaluated using the Guided Data Analisys. Adjusted means were obtained using the Least-squares means (LSMEANS) and treatments effects (I, CaI, E and L) were analyzed using the Tukey test. Response variables considered were cleavage, blastocyst, hatching, and DNA positive embryos rates. A probability value of P<0.05 was considered statistically significant. Results are reported as untransformed means +/- standard error of the mean (SEM).

RESULTS

Results show that the sperm fertilizing ability was not affected by protocols used for exogenous DNA-uptake as no significant differences on embryo development rates (cleavage, blastocyst and hatching rates) between experimental groups were observed (Table 1). Although the rate of positive embryo was constant among all groups we observed a low reproducibility for all methods. However rates of DNA-positive blastocysts did not differ among E, L, CaI and I groups (Figure 1).
The molecular analysis of blastocysts DNA revealed the presence of the plasmid sequence (Figure 2). No trace of target sequence was found at culture medium or washing medium (data not shown).

DISCUSSION

Results presented here show that DNA-uptake by bovine sperm occurs after electroporation, lipofection, CaI and incubation protocols with the same efficiency. Protocols used for exogenous DNA incorporation did not affect the fertilizing capacity of sperm as there was no significant difference in embryo development among experimental and control groups. Additionally, although the rate of positive embryo was constant among all groups we observed a low reproducibility for all methods. Therefore, other factors such as exogenous DNA sequence, size and concentration should be considered to improve SMGT [22].
SMGT is not yet fully established as a reliable genetic manipulation. Furthermore, is known that stable integration is rarely observed with SMGT protocols [23, 24]. One reason for the low efficiency may be related to endonucleases, which are activated according to the DNA concentration. This activity leads to exogenous sequences degradation or an apoptosis-like process in the sperm, significant decrease in spermatozoa motility eventually causing cell death [23, 25]. 
On the other hand, our group showed that in bovine species the exogenous DNA concentration had no apparent effect on endogenous DNA integrity and it could be due to the chromatin structure of sperm DNA [26]. Since bovine spermatozoa lack protamine 2 (only protamine 1 has been detected) and DNA fragmentation is more related to protamine 2, this could offer a better resistance to DNA damage to endonuclease activity compared to other species like murine and human. In the present study we used the same concentration of exogenous DNA as Feitosa et al. [26]. We can speculate that exogenous DNA concentration was not able to trigger endonuclease activity; hence, embryo development rates from all experimental groups were not different (p>0.05) from control group what suggests that endonuclease activity is not a barrier for SMGT in this species.
In another work from our group [27], it was reported that incubation time could reduce sperm viability causing acrosome membrane disruption during increased incubation periods. Our data is in accordance with Canovas et al. [28] that showed that after 5 min of incubation, more than 20% of bovine spermatozoa bind to exogenous DNA. Garcia-V�zquez et al. [29] described that exogenous DNA mainly bound to spermatozoa with reduced viability and as a consequence, only a low percentage of living cells were bound to DNA. According to these authors, frozen-thawed spermatozoa present a lower viability but a greater DNA-binding capacity when compared to fresh sperm cells, probably because the freezing process induces plasma membrane changes that facilitate the DNA-binding. Moreover, it is known that frozen semen have plasma membrane alterations similar to modifications occurred during sperm capacitation [30, 31]. Sperm DNA-binding may occur during this capacitation-like process, but since acrosome reaction may also take place prematurely, the sperm cell could die. This is a possible explanation for our results since the majority of oocytes could be fertilized by sperm without exogenous DNA, resulting in a low percentage of positive embryos with exogenous DNA.
We showed that all protocols for exogenous DNA-uptake by bovine spermatozoa can be used to produce transgenic livestock. Other methods could be used in order to produce transgenic farm animals such as direct microinjection of the egg pronuclei and viral-based constructs as vectors to introduce de foreign DNA into embryos. However, these methods show some disadvantages as (I) low efficiency since the zygote of most farm animals are opaque due to the lipid content in the cytoplasm, which makes the pronuclei visualization very difficult; (II) elevated costs and skills; (III) embryo manipulation at early stages of development; (IV) gene silencing by DNA methylation due to the presence of viral sequences and a high frequency of mosaicism in founder animals. Moreover, the use of retroviral vectors is affected by safety issues [32-34].
 The ability of transferring genes into animal genome is a prerequisite for further progress in animal trangenesis. However, the improvement of methods for genetic modification is needed. Key aspects to be improved include the DNA-uptake efficiency, the localization of the gene in the nucleus and gene targeting of the transgene in the genome. 

CONCLUSION
The results showed that all protocols for exogenous DNA-uptake are equally effective. All methods had a constant embryo production, despite the low reproducibility of exogenous DNA positive embryo rates. Therefore, other factors such as exogenous DNA sequence, size and concentration should be considered to improve SMGT.
 
ACKNOLEDGEMENTS
The authors would like to thank FAPESP for the financial support (03/08542-5). 

REFERENCES
Brusic V, Rudy G, Honeyman G, Hammer J, Harrison L (1998) Prediction of MHC class II- binding peptides using an evolutionary algorithm and artificial neural network. Bioinformatics 14: 121-130.

Niemann H, Kues W, Carnwath JW (2005) Transgenic farm animals: present and future. Rev Sci Tech 24: 285-298.
Kues WA, Niemann H (2011) Advances in farm animal transgenesis. Prev Vet Med 102: 146-156.
Eyestone WH (1994) Challenges and progress in the production of transgenic cattle. Reprod Fertil Dev 6: 647-652.
Robl JM, Wang Z, Kasinathan P, Kuroiwa Y (2007) Transgenic animal production and animal biotechnology. Theriogenology 67: 127-133.
Lavitrano M, Bacci ML, Forni M, Lazzereschi D, Stefano C, Fioretti D, et al. (2002) Efficient production by sperm-mediated gene transfer of human decay accelerating factor (hDAF) transgenic pigs for xenotransplantation. Proc Natl Acad Sci USA 99: 14230-14235.
Wall RJ (1999) Sperm-mediated gene transfer: advances in sperm cell research and applications. Transgenic Res 8: 313-315.
Arezzo F (1989) Sea urchin sperm as a vector of foreign genetic information. Cell Biol Int Rep 13: 391�404.
Wall RJ (2002) New gene transfer methods. Theriogenology 57: 189-201.
Gagn� MB, Pothier F, Sirard MA (1991) Electroporation of bovine spermatozoa to carry foreign DNA in oocytes. Mol Reprod Dev 29: 6-15.
Chen C, Smye SW, Robinson MP, Evans JA (2006) Membrane electroporation theories: a review. Med Biol Eng Comput 44: 5-14.
Rottmann O, Antes R, H�fer P, Sommer B, Wanner G, et al. (1996) Liposome-mediated gene transfer via sperm cells. High transfer efficiency and persistence of transgenes by use of liposomes and sperm cells and murine amplification element. J Anim Breed Genet 113: 401-411.
Smith KR (2002): Gene transfer in higher animals: theoretical considerations and key concepts. J Biotechnol 99: 1-22.
Ball BA, Sabeur K, Allen WR (2008) Liposome-mediated uptake of exogenous DNA by equine spermatozoa and applications in sperm-mediated gene transfer. Equine Vet J 40: 76-82.
Miao X (2012) Recent Advances and Applications of Transgenic Animal Technology, Polymerase Chain Reaction, Dr Patricia Hernandez-Rodriguez (Ed.), ISBN: 978-953-51-0612-8, InTech, DOI: 10.5772/38040. Available from: http://www.intechopen.com/books/polymerase-chain-reaction/recent-advances-and-applications-of-transgenic-animal-technology
Sim�es R, Nicacio AC, Binelli M, de Paula-Lopes FF, Milazzotto MP, et al. (2012) Sperm-mediated gene transfer: effect on bovine in vitro embryo production. Zygote 18: 1-5.
Zhang JJ, Muzs LZ, Boyle MS (1991): Variations in structural and functional changes of stallion spermatozoa response to calcium ionophore A32187. J Reprod Fertil Suppl 44: 199-205.
Perry AC, Wakayam T. Kishikawa H, Kasai T, Okabe M, et al. (1999) Mammalian transgenesis by intracytoplasmic sperm injection. Science 284: 1180-1183.
Pereira RJTA, Tuli RK, Wallenhorst S, Holtz W (2000) The effect of heparin, caffeine, and calcium ionophore A23187 on in vitro induction of the acrosome reaction in frozen-thawed bovine and caprine spermatozoa. Theriogenology 54: 185-192.
Parrish JJ, Susko-Parrish JL, Winer MA, First NL (1988): Capacitation of bovine sperm by heparin. Biol Reprod 38: 1171-1180.
Yamada C, Caetano HV, Sim�es R, Nicacio AC, Feitosa WB, et al. (2007) Immature bovine oocyte cryopreservation: comparison of different associations with ethylene glycol, glycerol and dimethylsulfoxide. Anim Reprod Sci 99: 384-388.
Tervi, HR, Whittingham DG, Rowson LE (1972): Successful culture in vitro of sheep and cattle ova. J Reprod Fertil 30: 493-497.
Sciamanna I, Piccoli S, Barveri L, Zaccagnini G, Magnano AR, et al. (2000) DNA dose and sequence dependence in sperm-mediated gene transfer. Mol Reprod Dev 56: 301-305.
Smith K, Spadafora C (2005) Sperm-mediated gene transfer: applications and implications. Bioessays 27: 551-562.
Spadafora C (2008) Sperm-mediated 'reverse' gene transfer: a role of reverse transcriptase in the generation of new genetic information. Hum Reprod 23: 735-740.
Spadafora C (1998) Sperm cells and foreign DNA: a controversial relation. BioEssays 20: 955-964.
Feitosa WB, Mendes CM, Milazzotto MP, Rocha AM, Martins LF, Sim�es R, et al. (2010) Exogenous DNA uptake by bovine spermatozoa does not induce DNA fragmentation. Theriogenology 74: 563-568.
Feitosa WB, Milazzotto MP, Sim�es R, Rovegno M, Nicacio AC, et al. (2009) Bovine sperm cells viability during incubation with or without exogenous DNA. Zygote 17: 315-320.
Canovas S, Gutierrez-Adan A, Gadea J (2010): Effect of exogenous DNA on bovine sperm functionality using the sperm mediated gene transfer (SMGT) technique. Mol Reprod Dev 77: 687-698.
Garc�a-V�zquez FA, Garc�a-Rosell� E, Guti�rrez-Ad�n A, Gadea J (2009) Effect of sperm treatment on efficiency of EGFP-expressing porcine embryos produced by ICSI-SMGT. Theriogenology 72: 506-518.
Harrison RA, 1997: Sperm plasma membrane characteristics and boar semen fertility. J Reprod Fertil Suppl 52: 195-211.
Leahy T, Gadella BM (2011) Sperm surface changes and physiological consequences induced by sperm handling and storage. Reproduction 142: 759-778.
Yang X, Tian XC, Dai Y, Wang B (2000) Transgenic farm animals: applications in agriculture and biomedicine. Biotechnol Annu Rev 5: 269-292.
Hofmann A, Kessler B, Ewerling S, Kabermann A, Brem G, et al. (2006) Epigenetic regulation of lentiviral  trasngene vectors in a large animal model. Mol Ther 13: 59-66.
Lavitrano M, Busnelli M, Cerrito MG, Giovannoni R, Manzini S, et al. (2006) Sperm-mediated gene transfer. Reprod Fertil Dev 18: 19-23.
Figure captions and table

Figure 1 � Percentage of DNA positive embryos produced by in vitro fertilization using different protocols for exogenous DNA-uptake by sperm cells (electroporation, lipofection, CaI, or incubation groups). Data presented as LSMEANS � SEM. 

Figure 2 - Electrophoresis on 1% agarose gel stained with ethidium bromide for detection of plasmid DNA. MW: DNA ladder; C+: plasmid DNA positive control, C-: PCR negative control (no sample); CE-: embryo from control group; I: embryos from incubation group; E: embryos from electroporation group; CaI: embryos from calcium ionophore group; L: embryos from lipofection group. White arrows indicate DNA-positive blastocyst

Table 1 � Embryo development rates (cleavage, blastocyst and blastocyst hatching) for in vitro fertilization using different protocols for exogenous DNA-uptake by sperm cells (E, L, CaI or I). Data presented as LSMEANS � SEM. 

UW_`mp��������		
	!	"	9	:	_	a	w	x	y	�	�	���̿�������������������wj_Ph%�h~FfHq�
����h%�h~FB*phh%�h~FB*H*phh%�h~F5�B*H*ph)h%�h~F5�B*fHph"""q�
����h
^�h%�B*H*phh
^�ht�B*H*phh
^�ht�B*phh
^�ht�5�B*phDDDh
^�ht�5�B*phh%�ht�B*mH	phDDDsH	h%�ht�mH	sH	h%�ht�5�mH	sH	Wa	x	
�
(s����������������������������������
$d�a$gd��
$d�a$gdt�$dh7$8$H$a$gd~F$d�7$8$H$a$gd%�
$d�a$gd%��	


p
q
�
�
��<=���̽����uj�uQ6u5h
^�h%�0J*5�B*fHmH	phq�
����sH	1h
^�h%�5�B*fHmH	phq�
����sH	h%�h%�B*phh%�h%�B*mH	phsH	 h%�h%�5�B*mH	phsH	 h~FB*fHphq�
����h
^�h�
?B*phh�
?B*mH	phsH	h%�h~FB*mH	phsH	 h%�h~FB*H*mH	phsH	h
^�h~FB*mH	phsH	%h
^�h~FfHmH	q�
����sH	������������



'
,
2
�˲����vj[I<1<1<1<h%�h-X�mH	sH	h%�h-X�0JmH	sH	#h%�h-X�5�B*\�mH	phsH	h
^�h��B*mH	phsH	h��B*mH	phsH	h�
?B*mH	phsH	h
^�h~FB*mH	phsH	h
^�h%�B*mH	phsH	#h
^�h~F5�B*H*mH	phsH	1h
^�h%�5�B*fHmH	phq�
����sH	5h
^�h%�0J*5�B*fHmH	phq�
����sH	1h
^�h%�5�B*fHmH	phq�
����sH	2
�
�
�
�
�
�
�
�
�
�
�
�
�
�
�
�
ad��C_��,*3���-.�����������������������̬̝̝̽̽̎�ۂz��۝�nh-X�B*mH	phsH	h
^�mH	sH	h
^�h
^�6�mH	sH	ht�h[�B*mH	phsH	ht�h
^�B*mH	phsH	 ht�h-X�6�B*mH	phsH	ht�h8ECB*mH	phsH	ht�h-X�B*mH	phsH	ht�h-X�mH	sH	ht�h8EC0JmH	sH	ht�h-X�0JmH	sH	)����)x}
{�!"#����qz�nou	
Y������������������������q�e������Y���ht�h-X�6�mH	sH	h��B*mH	phsH	#ht�h-X�6�B*]�mH	phsH	h@umH	sH	ht�h-X�mH	sH	h@uB*mH	phsH	ht�h%�B*mH	phsH	ht�h-X�B*mH	phsH	#ht�h-X�5�B*\�mH	phsH	h��B*mH	phsH	ht�h��B*mH	phsH	#ht�h��5�B*\�mH	phsH	"()�w���!!"!7!8!A$B$m$�&�&�&�'�'�')),)�*������������������������$��d�7$8$H$`��a$gdt�$��d�`��a$gdt�
$d�a$gdt������#��
��#���� � "!7!c!k!�"�"�"�"�"�#�#�#�#�#�#������������������|�m�m�|�\�| ht�h-X�B*H*mH	phsH	ht�h�bB*mH	phsH	#ht�h-X�6�B*]�mH	phsH	#ht�h-X�5�B*\�mH	phsH	ht�h8ECB*mH	phsH	!ht�h8EC0JB*mH	phsH	!ht�h-X�0JB*mH	phsH	h@umH	sH	ht�h-X�mH	sH	h@uB*mH	phsH	ht�h-X�B*mH	phsH	!�#A$m$�$�$V%Z%�%�%�&�&�&�&�'�'�'�'�(�(�(�(),)2*8*q*r*z*{*�*�*++++>+Y+��ϾϲϾϡϾϐϡϐ�ϡ�sϐ�ϡϐ��ght�h-X�6�mH	sH	h@uB*mH	phsH	 ht�h-X�B*H*mH	phsH	!ht�h-X�B*PJmH	phsH	 ht�h-X�6�B*mH	phsH	hd~2B*mH	phsH	 ht�h-X�B*H*mH	phsH	ht�h-X�B*mH	phsH	#ht�h-X�6�B*]�mH	phsH	ht�h'~B*mH	phsH	$�*�*�*=+>+Y+�/�/0�2�23t5�6�6�9;;;�<�<�<????�������������������������$��d�`��a$gdt�
$d�a$gdt�Y+�+�+�,�,n-p-�-�-�-�-T.V.�.�.�/�/�/000�1r2s2�2�2�2�2�2�23�3�3�4�4t5�5�5�5�5�5�6�6�����������������������|�k�|!ht�h-X�B*PJmH	phsH	#ht�h-X�6�B*]�mH	phsH	 ht�h-X�B*]�mH	phsH	ht�h�bB*mH	phsH	 ht�h-X�B*H*mH	phsH	ht�h-X�B*mH	phsH	ht�h-X�6�mH	sH	ht�h-X�H*mH	sH	ht�h-X�h@umH	sH	ht�h-X�mH	sH	*�6|7}7S8T8�8�8$9%9@9A9_9`9;;a;|;�;�;�;�;<9<�<�<??#@�@�A�A)C#D'D)D�D�D����������������𫚈�}��}u}d}!ht�h-X�0JB*mH	phsH	h�lmH	sH	ht�h-X�mH	sH	#ht�h-X�5�B*\�mH	phsH	 ht�h-X�B*\�mH	phsH	#ht�h-X�6�B*]�mH	phsH	 ht�h-X�B*H*mH	phsH	 ht�h-X�B*H*mH	phsH	!ht�h-X�B*PJmH	phsH	ht�h-X�B*mH	phsH	$?�@�A�A�A�A)DFQIDN�Q�R�R�R5T7TGT�T�T������������������$d�-DM�
����a$gdt�#$��d���`��a$gdt�#$��d�`��a$gdt�
$d�a$gdt�$��d�`��a$gdt�#$���d�]�`��a$gdt��D�D�D�E�E�E�E�F�F�F�F*H,H-H.HpIrIsItI0J2J3J4J�J�J�J�J�L�L�L�L�Q�Q�Q�Q�Q�Q�Q�R�R�R�S5T7TGT�T�T�T�T���������������������������ޤޕޤht�h-X�B*mH	ph"""sH	#ht�h-X�5�B*\�mH	phsH	h�L�mH	sH	h�lmH	sH	hd~2B*mH	phsH	h�lB*mH	phsH	ht�h-X�B*mH	phsH	ht�h-X�mH	sH	hd~2mH	sH	0�T�TeUfU�U.V�V"W&X�XYRY�YQZ`[�[�\�]�^6_�_�`8a����������������������
$
&Fd��gdOz�
&Fd�gdOz�$
&Fd��d�d*$-DM�
����[$\$a$gdk�
$d�a$gdt��TIUeUfU�U�U�U�U�U�U�U�U�U�U�U�U�U�UVV V#V$V:V;V?V@V�V�V�V�V�V�V�V�V�VW�ɺ����������u�������������f��f�����h
^�hOz�B*mH	phsH	hOz�B*mH	phsH	hk�hOz�B*mH	phsH	h��hOz�B*mH	phsH	hk�B*mH	phsH	ht�hOz�B*mH	phsH	ht�h-X�B*mH	phsH	1hk�hk�B*CJKHOJQJaJnHphQQQtH9hk�hk�B*CJKHOJQJaJmH	nHphQQQsH	tH$WWWWWhWnWoWpWtWuW�WXXX&X.X/X3X4X�X�X�X�X�X�X�X�X�X�X�X�X�XYYYYY�����ƺ�����ƫ����������ƜƜ�{lƜ`�hBB*mH	phsH	hk�hBB*mH	phsH	#ht�hB6�B*]�mH	phsH	h�
?hBB*mH	phsH	ht�hBB*mH	phsH	h
^�hOz�B*mH	phsH	hOz�B*mH	phsH	hk�B*mH	phsH	hk�hOz�B*mH	phsH	ht�hOz�B*mH	phsH	h�9�hOz�B*mH	phsH	%YYYY6YDYEYGYHYQYRYqYrYvYwY�Y�Y�Y�Y�Y�YZZZ3ZFZGZIZJZQZ�Z�Z�Z�Z�Z������������䐮����rfW���ht�hOz�B*mH
phsH
ht	B*mH
phsH
ht	hOz�B*mH
phsH
h�
?hOz�B*mH	phsH	ht	hOz�B*mH	phsH	h��hOz�B*mH	phsH	ht	B*mH	phsH	hOz�B*mH	phsH	hk�hOz�B*mH	phsH	h�9�hOz�B*mH	phsH	ht�hOz�B*mH	phsH	hk�B*mH	phsH	"�Z�Z?[@[A[Q[R[U[V[`[i[j[n[o[p[�[�[�[�[�[�[�[�[�[k\w\x\z\{\�\�\�\�\�]^^^ ^$^%^q^�������䯧�������������~��������s�s����h
^�hOz�mH	sH	h�
?hOz�mH	sH	ht	hOz�mH	sH	h�9�hOz�mH	sH	hOz�mH	sH	ht	mH	sH	ht�hOz�mH	sH	ht	hOz�B*mH	phsH	hOz�B*mH	phsH	h�9�hOz�B*mH	phsH	ht�hOz�B*mH	phsH	ht	B*mH	phsH	(q^w^x^z^{^�^�^�^�^�^___(_)_+_,_6_G_j_p_q_r_v_w_�_�_�_�_�_�_�_�_`�`������������ܬ蠑������t�e������ht	hOz�B*mH	phsH	 h�9�hOz�B*]�mH	phsH	ht	B*mH	phsH	ht�hOz�B*mH	phsH	hOz�B*mH	phsH	ht	\�mH	sH	h�9�hOz�5�mH	sH	h�9�hOz�mH	sH	hOz�mH	sH	ht	mH	sH	ht	hOz�\�mH	sH	ht�hOz�mH	sH	h�9�hOz�]�mH	sH	"�`�`�`�`�`�`�`�`�`�`a(a)a+a,a8apavawaxa|a}abbbbbDbEbIbJbKb�b�b�b�b�b�b�b�b�b�b�b�b�b+c9c:c�������÷������è����������÷��ω��������x� h
^�hOz�B*]�mH	phsH	ht	B*\�mH	phsH	 h�9�hOz�B*]�mH	phsH	h
^�hOz�B*mH	phsH	hOz�B*mH	phsH	ht	B*mH	phsH	 ht	hOz�B*\�mH	phsH	ht�hOz�B*mH	phsH	 h��hOz�B*]�mH	phsH	/8ab�bGc�cXd�dwe#f�f�gh�h3i�icj~jjokpkm��������������������$�n���dh^�n`���a$gd��$�n���d�^�n`���a$gd��
$
&Fd��gdOz�
&Fd�gd�L�
&Fd�gdOz�:c<c=cGc\c]cacbc�c�c�c�c�c�c�c�c�c@dJdKdMdNdWdXdddedidjd�d�d�d�d�d�d�deeeee[eaeiejele�������Ŵ������Ŵ��Ś����Ŵ���������Ō{�� h
^�hOz�B*]�mH	phsH	ht	B*]�mH	phsH	hOz�B*mH	phsH	ht	B*\�mH	phsH	 h�9�hOz�B*]�mH	phsH	ht�hOz�B*mH	phsH	h
^�hOz�B*mH	phsH	ht	B*mH	phsH	 ht	hOz�B*\�mH	phsH	,lemewe�e�e�e�e�e�efffffHfIfMfNfOf�f�f�f�f�fggg g�g�g�g�g�g�g�g�g�g�g�g�ghh
hhh)h*h��������¶ª����͢¶ª�����–ª�¢¢��–����w�ht�h�L�mH	sH	h��hOz�mH	sH	ht	\�mH	sH	h��hOz�]�mH	sH	hOz�mH	sH	ht	hOz�\�mH	sH	h�
?hOz�]�mH	sH	ht�hOz�mH	sH	ht	mH	sH	h
^�hOz�mH	sH	h
^�hOz�B*mH	phsH	 ht	ht	B*\�mH	phsH	.*h.h/h�h�h�h�h�h�h�h�h�h�h�hi&i'i(i)ijipiqiriviwi�i�i�i�i�i�ij j!j"j&j'j(jFjWjXjZj[jcj����������ʶʪ�ʢʢʢʖʊ�����ʖʊ��h
^�hOz�mH	sH	h�v~hOz�\�mH	sH	h��hOz�]�mH	sH	h�v~mH	sH	ht	hOz�\�mH	sH	h�9�hOz�]�mH	sH	hOz�mH	sH	ht�hOz�mH	sH	ht	h�L�\�mH	sH	h��h�L�]�mH	sH	ht	mH	sH	ht�h�L�mH	sH	+cjdj}j~jj�j�j�jnkpk{kmm!m�Ͳ��vjvbSH=2hthh��mH	sH	h��h��mH	sH	h�tqh��mH	sH	h�tqh��B*mH	phsH	h��mH	sH	hkh��6�mH	sH	hkh��mH	sH	hkh��B*mH	phsH	Bh�v~h��0J*B*CJOJQJaJfHmH	phQQQq�
����sH	5h�v~h��0J*5�B*fHmH	phq�
����sH	1h�v~h��5�B*fHmH	phq�
����sH	1h�v~h�L�5�B*fHmH	phq�
����sH	
mm�m�m�� �+�;�D�T�����������$d�$Ifa$gd��$��� �d�^��`� �a$gd��dhgd��
!m3mmmum�m�m�m�T�U������������ �U�V�W�X�Z�[�]�^�`�a�c�d�{�|�~�����������������ü����弱�����������������hPih�v~mHnHuht�jht�Uh�\`jh�\`Uh��h-X�mH	sH	hthh��hthh��5�mH	sH	Uh��mH	sH	hthh��6�mH	sH	hthh��mH	sH	hthh��B*mH	phsH	%GROUPSCLEAVAGE
(LSmeans � SEM)BLASTOCYST
(LSmeans � SEM)HATCHING
(LSmeans � SEM)CaI63.84 � 3.8928.07 � 4.4332.32 � 9.72Electroporation65.53 � 3.5926.61 � 4.9841.72 � 9.38Incubation65.02 � 5.2423.95 � 3.0038.30 � 9.88Lipofection57.22 � 3.4527.41 � 5.2028.82 � 8.67Control group67.55 � 5.5521.76 � 2.9231.57 � 8.44








 PAGE   \* MERGEFORMAT 17




T�U�Y�f�s���qbSSS$d�$Ifa$gd��$d�$Ifa$gd���kd$$If�l�\�A� �����������4�4�
la�lp�(��������yt4������������qbSSS$d�$Ifa$gd��$d�$Ifa$gd���kd�$$If�l�\�A� �����������4�4�
la�lp�(��������yt4����ĨѨި�qbSSS$d�$Ifa$gd��$d�$Ifa$gd���kd�$$If�l�\�A� �����������4�4�
la�lp�(��������yt4�������qbSSS$d�$Ifa$gd��$d�$Ifa$gd���kdR$$If�l�\�A� �����������4�4�
la�lp�(��������yt4� �.�;�H�U�qbSSS$d�$Ifa$gd��$d�$Ifa$gd���kd$$If�l�\�A� �����������4�4�
la�lp�(��������yt4U�V�W�Y�Z�\�]�_�`�b�c�qfa_a_a_a_gdt�
$d�a$gd���kd�$$If�l�\�A� �����������4�4�
la�lp�(��������yt4
c����������������
$d�a$gd��(($a$?0P0p�1�F:p�NZ��. ��A!��"��#��$��%��������$$If�l!vh5��5��5��5��#v�#v�#v�#v�:V�l��5��5��5��5��/�a�lp�(��������yt4�$$If�l!vh5��5��5��5��#v�#v�#v�#v�:V�l��5��5��5��5��/�a�lp�(��������yt4�$$If�l!vh5��5��5��5��#v�#v�#v�#v�:V�l��5��5��5��5��/�a�lp�(��������yt4�$$If�l!vh5��5��5��5��#v�#v�#v�#v�:V�l��5��5��5��5��/�a�lp�(��������yt4�$$If�l!vh5��5��5��5��#v�#v�#v�#v�:V�l��5��5��5��5��/�a�lp�(��������yt4�$$If�l!vh5��5��5��5��#v�#v�#v�#v�:V�l��5��5��5��5��/�a�lp�(��������yt4^+���������666666666vvvvvvvvv666666>666666666666666666666666666�6666666666�666666666666hH66666666666666666666666666666666666666666666666666666666666666666�62���� 0@P`p������2(�� 0@P`p������ 0@P`p������ 0@P`p������ 0@P`p������ 0@P`p������ 0@P`p��8X�V~_HmHnHsHtHH`�HNormal*$CJKH_HaJmHsHtH>A`�>Fonte par�g. padr�oXi@�X
0
Tabela normal :V�4�4�
la�,k �,
0	Sem listaD�o��DDefault Paragraph Font>��>Texto de bal�o Char@��@annotation referenceH��!HTexto de coment�rio CharL�"1LAssunto do coment�rio CharB��ABCorpo de texto 3 Char0��Q0medium_text1,�O�a,
long_text1FU�qF	HyperlinkB*mH�ph�sH�>*_H�tH��O��hpsP���PRecuo de corpo de texto CharN��NT�tulo1
���x$OJQJCJPJ^JaJ@B�@Corpo de texto
��x*/��*Lista^JJ��JLegenda1
�x�x$CJ6^JaJ]0��0�ndice$^J8��8List Paragraph4�4Balloon Text :�:annotation text!@�"@annotation subject"2�O22Body Text 3#^C@B^Recuo de corpo de texto$^�]�`���x6(�Q6
�q%0N�mero de linha:b:
't�0	Cabe�alho
&
��8!D��qD&t�0Cabe�alho CharCJKHaJtH4 @�4)t�0Rodap�
(
��8!>���>(t�0Rodap� CharCJKHaJtHB�O��B~Fapple-converted-spacePK!����[Content_Types].xml���j�0E����ж�r�(��΢Iw},��-j��4	��w�P�-t#bΙ{U�����T�U^h�d}㨫��)��*1P�'��	�^��W��0)��T�9<�l�#��$yi}��;�~@��(��H����u�*Dנz��/0�ǰ��$��X��3aZ����,�D0j~�3߶�b��~i>���3�\`�?�/�[���G��\�!�-�Rk.�s�Ի�..���a濭?��PK!�֧�6_rels/.rels���j�0���}Q��%v/��C/�}�(h"���O�
����=������ ����C?�h�v=��Ʌ��%[xp��{۵_�Pѣ<�1�H�0���O�R�Bd���JE�4b$��q_����6L�R�7`������0̞O��,�En7�Li�b��/�S���e��е�����PK!ky���theme/theme/themeManager.xml�M
� @�}�w��7c�(Eb�ˮ��C�AǠҟ����7��՛K
Y,�
�e�.���|,���H�,l����xɴ��I�sQ}#Ր���� ֵ+�!�,�^�$j=�GW��)�E�+&
8���PK!��.��atheme/theme/theme1.xml�YMoE�#�F{om'vGu�رh�F�[��xw�;���jf��7���q�7��J\ʉ�(�"�/����z'�4IA�!��>�Ǽ3�|�^��>���5.�=D�4	;ޭ��ª���I�OHǛ�]Y���xME$&���;^�T�V�I����S���	1V�(�Z ��Ym�^_�Ř&JplG@��nN&�'�zξ�@F��^�j�$��K_P��A�!�&g���Ǭへ���=�!����n>^m�r
�eDLC[��OF�{KF�Dž�Ơپ�Y�7�q�~��o��>�ku)�lVݜg	d�.���[���/�_^й��v[�L�Ԁ���~����Xr�d�|����8x�����R{���
(b4�[@��2�l��
��z�� ��"&<Q�L���b(�fX��YJ&؇���x,(�R���7vɗKZ �I����a��:�^>���'����?>xpx�G�ȡ��IX�z��g=��������2��>���ϫ�PCsu����������=��o<.�G4&� h��`��9��Q�"L�I(q���
�}9�3̲�8zt���zH����0SE+$_�b��9�rQ�kZV�ͣiV�2n�*�=�8��OS�yZ:��"⨹�p�pH��~������_��/����P�Ŵ�%#:v�iN�Ec�ˬ�f����ۨ�Y�՛d�EBU`V���0ǍW�TḊ�Ǭ���XEUJg�/�RA�C�8�D�*���-���U�m6�]�Pt���u�y��z��*�&Q��܃�h��*�6w+D?Cprl�oS��np���J��o�B���Ӂc���3
����ch�Ͽ~T�Yok#ހ=�������pG�n�����=wO�i���k��Z���o����i�B��s���͜�zL�PƆj��ui&e	�E0�EMl���8;�|͚��64Hp�U�0�)L�
O3	e�:�(��xf����ä������)H��y`���r~B(ؘ-'4g�\вfpZa˗2�`��kh�N-�aT3�ΑV��\4
o��`v/���]���	f$�~�p����p@�i�c�0A�s�\@�T�H�N�ZIZ[�}i�	RY\�qy��$Jyϣ���H9��\�,A��Zjy��iǛ����)D]��n�|%lڟX̦���l熹EЀ���>�
�6��lj�WY
�DK��/����e�����by��_��膖L&�W�`�V���c�J�T1��4fS��!�:U���J��0A?�����y�6���Xg�1K#��[]�y%[���B�TRl���wvSLɟ�)�4������.Xt|���z�x\��CJ#�L�w@���,�����e�_�}��֜�a�N}j��HP؏T$ف�d��f�l�,Y��dTI]�Z��d����+zo�P�n�I��h���Y�C=����!��kk�|l1�Qn6M�BŊ]���|�-�_�Ǭf^ �����Mθ�ڎ�`�R+W��h1,Q
�>H����
��*�:��[�䠙A�@V_���
�.�ap��6�4+��lt�^�7�s�t�G��5;M����b8s�9�x���<�ڮ�j�����I~�1�1�q�����M��2%M2��K��94u�o%����PK!
ѐ��'theme/theme/_rels/themeManager.xml.rels��M
�0���wooӺ�&݈Э���5
6?$Q��
�,.�a��i����c2�1h�:�q��m��@RN��;d�`��o7�g�K(M&$R(.1�r'J��ЊT���8��V�"��AȻ�H�u}��|�$�b{��P����8�g/]�QAsم(����#��L�[������PK-!����[Content_Types].xmlPK-!�֧�6+_rels/.relsPK-!ky���theme/theme/themeManager.xmlPK-!��.��a�theme/theme/theme1.xmlPK-!
ѐ��'�	theme/theme/_rels/themeManager.xml.relsPK]�
<?xml version="1.0" encoding="UTF-8" standalone="yes"?>
<a:clrMap xmlns:a="http://schemas.openxmlformats.org/drawingml/2006/main" bg1="lt1" tx1="dk1" bg2="lt2" tx2="dk2" accent1="accent1" accent2="accent2" accent3="accent3" accent4="accent4" accent5="accent5" accent6="accent6" hlink="hlink" folHlink="folHlink"/>e�����	+++.�	�2
���#Y+�6�D�TWY�Zq^�`:cle*hcj!m��79:;<>?ABDFGHIJKMNOPR�*?�T8amT�������U�c���8=@CELQUVWXYZ[$'.!����8�@�����������0�(	�
��B
�S����	?���	OLE_LINK2	OLE_LINK1_GoBack�9�94J�e#:#:4J�eW^x�������<Aaijw����������4=_ns~��		�	�	�	
�����	�������HWYh��!*��w�����=@����u���IP��FK������u������<KP[������FU����������� !%!'!*!�!�!�!�!""-"1"$#$�$�$�$�$�$�$�$%5%=%x%�%�&�&�&�&�&�&''$'/'_'a'b'l'P(W(�(�(1)4)k)t)�)�)?*D*!+$+)+3+t++�+�+�-�-�-�-�-�-�-�-�-�-�-�-.... .*.k.s.�.�.�.�.S0X0t0~0�0�0�0�0k1t1}1�1&282I2S2{3�3�3�344D4N4�5�5�6�6�6�67 78 8"8-8/828;;�<�<=
=I=R=�=�=�=�=>">z>�>??!@(@�@�@�C�C�F�F!G*G�G�G�H�H�J�J�J�JIKWKfKmKqKuKyK�K�K�K�K�K�K�KLLLL�L�L�L�L�L�L�L�L�L�LMM"M+M/M4M9M>MBMMM\MdM�M�M�M�MNNNN
NN�N�N�N�N6ODOROWO\OcOgOmOxO�O�O�O�O�O�O�OPP7P;P@PFPQPYPfPkPoPuPyPP�P�PQQ!Q'QAQEQ�Q�Q�Q�Q.S4S�S�S�S�S�S�S	TT�T�TU
UUUU"U#U(U@UGUKUTU�U�U�U�U�U�U.V7V�V�V�V�V�V�V�V�VW!W"W(WNWTWXW_WdWkW�WXXXX
XXXX$X*X5X:X@X�X�X�X�X�X�X�X�X�X�X�X�X�X�X/Y5YPYYY�Y�Y�Y�YDZJZXZaZ�Z�Z�Z�Z�Z�Z�Z�Z[[i[w[~[�[�[�[�[�[�[�[�[#\*\8\<\@\E\�\�\�\�\�\�\]]]]�]�]�]�]�]^^^^%^�^�^___"_I_Q_U_^_b_f_�_�_�_�_�_�_�_�_�_�_````F`L`M`S`X`Z`a"a$a/a1a4a�a�a�a�a�b�b�b�b�b�b�b�bccEcOcTc^c�c�cdd'd.d@dGdPdSd|d�d�d�dReTeUeWeXeZe[e]e^e}e�egoawxy�������nu��=@�������'!*!�3�3�6�6/828�K�K�L�LOOVV�^�^F`b`1a4a�b�b�c�cReTeUeWeXeZe[e]e^e}e�e33333333333333333333333	Y�#�#*>0>IKfK�K�KL.L�L�LM"M�M&N�N�N�NO6ORO�O�O3PQP?Q`Q�Q�QkR�RqT�TU6U�U�U�V�VW8WXX�X�X+YGY�Y�Y@ZXZ�Z�Z[[w[\#\�\�\�]�]�]^�^�^_3_�_�_!`F`b`�cQeReTeUeWeXeZe[e]e^e}e�eR��������������`l�̏���������h
�����^��`����h�H.�h
�����^��`����h�H.�h
�p�L�^�p`�L��h�H.�h
�@���^�@`����h�H.�h
����^�`����h�H.�h
���L�^��`�L��h�H.�h
�����^��`����h�H.�h
�����^��`����h�H.�h
�P�L�^�P`�L��h�H.�������^��`���.��������^��`���.��p����p^�p`���.��@����@^�@`���.������^�`���.��������^��`���.��������^��`���.��������^��`���.��P����P^�P`���.R��`l������������YM�g$�#~Ft	B'~@,r�b4�q%d~2�
?8EC�NZ�\`Pi�ln@u�v~[�
^��L���Oz��_�<����9�-X�t�8}�k�%��"���ReTe�@�[`[`��[`[`@$%&�cex@x,x.x`@xP@��Unknown������������G��*�Ax�	�Times New Roman5��Symbol3.��*�Cx�	�Arial9�SymbolMTA.���Trebuchet MS;���(SimSun�[SO5.��.�[`�)�TahomaA���� B�Cambria Math"A� �h0m�r',�~U3����~U3�! ����20dee
3�Q ��X����������������������B�!xx���RenataRenata����Oh��+'��0h�������	��
$0
<HPX`�RenataNormalRenata5Microsoft Office Word@�%@@8)�n��@v������~U����՜.��+,��0�hp��������
���Hewlett-Packard Company�3eT�tulo	

 !"#$%&'()*+,-./0123456789:;<=>?@ABCDEFGHIJKLMNOPQRSTUVWXYZ[\����^_`abcd����fghijklmnopqrstuvwxyz{|}~���������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������Root Entry��������	�F�c/������Data
������������]1Table����e�9WordDocument����A�SummaryInformation(�������������DocumentSummaryInformation8���������CompObj������������}������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������
����	�F+Documento do Microsoft Office Word 97-2003
MSWordDocWord.Document.8�9�q

Youez - 2016 - github.com/yon3zu
LinuXploit