<%BANNER%>

Strategies to Enhance Fertility in Dairy Cattle during Summer including Use of Cryopreservation of in Vitro Produced Embryos

xml version 1.0 encoding UTF-8
REPORT xmlns http:www.fcla.edudlsmddaitss xmlns:xsi http:www.w3.org2001XMLSchema-instance xsi:schemaLocation http:www.fcla.edudlsmddaitssdaitssReport.xsd
INGEST IEID E20110218_AAAADW INGEST_TIME 2011-02-18T22:31:53Z PACKAGE UFE0013409_00001
AGREEMENT_INFO ACCOUNT UF PROJECT UFDC
FILES
FILE SIZE 6434 DFID F20110218_AACNQA ORIGIN DEPOSITOR PATH franco_c_Page_095thm.jpg GLOBAL false PRESERVATION BIT MESSAGE_DIGEST ALGORITHM MD5
d9a4c427ef71316c383c7c5eb51b34a9
SHA-1
88ab5ab8ec532b1d19cb9c3e89a099ca25b5a4ab
3304 F20110218_AACNPM franco_c_Page_080thm.jpg
0eba6432386089378499fc4078293656
612b10e7776ca8edc87a03ee263d3e4e873f0bec
5124 F20110218_AACNOX franco_c_Page_062thm.jpg
1a1c9fe336d9f752fc44c9dfaf2e91b9
ebbda0fc4b45ed6773ff92bd82de855d63d28f98
8423998 F20110218_AACMMK franco_c_Page_004.tif
ad0f294b6649e8f6f3c64b1f9373084d
31f2017dc0ce29ae6b899e99b0b2f8e43ed2ad6b
F20110218_AACMLV franco_c_Page_028.tif
8b4f54fadcf074e177f48987fe4ff8a8
a5e75d4637f23571b05609bdfdb87efcaab7f346
6125 F20110218_AACNQB franco_c_Page_096thm.jpg
0f2e5f294f3f74a5d44adf9c853ffe24
9c88abeafd630d03c454149dc148881b1b7a8fc6
3553 F20110218_AACNPN franco_c_Page_081thm.jpg
35e9868a475e70a37c91acea0734343f
8dddd775d68ae80da45b2668001dad4e872838e9
2925 F20110218_AACNOY franco_c_Page_063thm.jpg
ba8383cb1f6f0905772d93862584ce1d
9a150f94ee5eaaf7de9d02787e0805f86543f51d
F20110218_AACMML franco_c_Page_006.tif
d1ab5ed22adbd0ad96c8ce362fac239a
339251287ecd44195e6939f595f797ff204659b9
63285 F20110218_AACMLW franco_c_Page_128.pro
56b49748e4a9ebd5a5c3e7c02e0400ec
14eae048fa9fa753af8ae05d842ed04b716f6fc7
5887 F20110218_AACNQC franco_c_Page_097thm.jpg
51085b254900ea77f09b8c0130e26c3f
fe450a501bdc8e9c77705c56cefb68cb99fece47
3958 F20110218_AACNPO franco_c_Page_082thm.jpg
9688db529e8809809a09642d0090883b
3a0d9a024986113a946a7adf0b79be98a4364fc5
5729 F20110218_AACNOZ franco_c_Page_064thm.jpg
4f8beb5177521899707f63a63c59a5f1
82163c57b4f149d0e7f6baeb4e3adaab1f68af85
F20110218_AACMMM franco_c_Page_007.tif
191a7c6decd1b5b184f2a9809982b26e
142fb49c4db794e068f26f8845cdefc35707b63f
1051971 F20110218_AACMLX franco_c_Page_091.jp2
88fbd1c0aa79f7890f74878b77f2ae20
59f703744e8ec981fa944d1c2c1844d911a1bf8c
F20110218_AACMNA franco_c_Page_023.tif
7461c7c18c7da12daa0fbcf4fd5b2625
847988564858b9e728a73282125ff2e489e1dd6d
6096 F20110218_AACNQD franco_c_Page_098thm.jpg
d3228ac475a9fa18062a6fef0fa46572
4fdb20aabb606e89dcbb7b813185b2b67fcb34e5
3020 F20110218_AACNPP franco_c_Page_083thm.jpg
0daa724d65f27e4ac7f5fee4e73488ff
7ec826d991cfef79a223d1267c2ed1348d562aa1
F20110218_AACMMN franco_c_Page_008.tif
319fa759b88bf414565cc29607f96713
eb1ed7d792aced27e82db57ad0ed2b3f836d1f52
81368 F20110218_AACMLY franco_c_Page_091.jpg
4a241f3f842667ab7ea2bf2ae7c9a6a5
ac45a7cde8e5f914633e731ead1fa92211e77038
F20110218_AACMNB franco_c_Page_024.tif
e81bca685c4b95ecf433c7f449f66793
d61f4c9169c08f74b3ece333fb30c199284d8e1c
6808 F20110218_AACNQE franco_c_Page_104thm.jpg
643bc5f130f4ab318a5c6c316a365155
ced9f0ccfdcd529781d6660e13f5668febe3e7ac
5640 F20110218_AACNPQ franco_c_Page_084thm.jpg
c22b0327651ad7b95713b42222a69f5c
0e9ad5bf85afe8ae87fa93c13d35456254a1e574
F20110218_AACMMO franco_c_Page_009.tif
7db05fdd34bc3fad36d974b1d3ab8820
8ff5eb4e90e89c29ad3dea9509b23d91777fb8c9
F20110218_AACMLZ franco_c_Page_005.tif
9965bab88f60c303b052bf213a4fc4bb
401bb02394df575df532740c0cf49343f6969e8a
F20110218_AACMNC franco_c_Page_025.tif
795281fcf2bed88f8b2fed145c928313
bf0253b9902530429a01b547588b7b52376bc876
6699 F20110218_AACNQF franco_c_Page_105thm.jpg
6fbc7eeb533e697bd719d0d119c6a245
8e5ea4e2b5b67fce56788d24b7d1c5be2b9c09e0
5954 F20110218_AACNPR franco_c_Page_085thm.jpg
48e87973a45a0cf3342cd40f98b873c6
a8417e4a18fc4420523923110c42e3583f4a9391
F20110218_AACMMP franco_c_Page_010.tif
bd472dc1bb4e539a0fb44869e49ba84a
61e1b59c8a6f935d52ee640f0938ec54d3b2e18c
F20110218_AACMND franco_c_Page_026.tif
9fe43584f297e019ef6bc367d8819a18
f904135d78888f0fe5ab9f6e41f4cfa97f8c8ad8
6488 F20110218_AACNQG franco_c_Page_106thm.jpg
cbc5fc177c1212e3018a218e7f92c958
f1e8089f9a1df37f8fac9dbc90a92eb1a21556e3
F20110218_AACMNE franco_c_Page_027.tif
ecbeb5498894c8f4ad1efe45cfac1bc7
323916aad144a142626dd254a44c84f70f21a110
6516 F20110218_AACNQH franco_c_Page_107thm.jpg
a09956d1c73eb6a2e85614d567b7b4f6
bf49c6f57572587ee0da5dd1bd4b158e9669ae1f
6343 F20110218_AACNPS franco_c_Page_086thm.jpg
7b079dd2804bba73c9b2cea97359c5ea
f968f3e08f2b129bc923d34b6ec19b785708c8c4
F20110218_AACMMQ franco_c_Page_011.tif
42c1bd278a982a4e4bee893016904924
d033471b6fd3d470527b0f8048c7469f6ad604ce
F20110218_AACMNF franco_c_Page_029.tif
23c663d4a502b454718d8bb2c108c6f3
043320166ccb4e1415d4b7759285e6a450da858a
6560 F20110218_AACNQI franco_c_Page_108thm.jpg
fd383859df2328deaf619bbcafe44786
8c402b9642bcf297d8d79cd80c7858dce031c4c9
6413 F20110218_AACNPT franco_c_Page_087thm.jpg
a43400fb71f50857841e03e067671572
4d1c95ebab49109ac76dbd34b5f516fa44e41506
F20110218_AACMMR franco_c_Page_012.tif
a9046c94192d5003247b61bf67beb1bd
cb2209a3e74705e2dd69128a9d02ffe8051e6f9e
F20110218_AACMNG franco_c_Page_030.tif
6669934f0cc6d0ec0ab623e0074cc519
81be3f3df2ff69fd1aa81e8a9084c5efed1942c1
6446 F20110218_AACNQJ franco_c_Page_109thm.jpg
943b2d7cb72dd06b30ff18df29f028b2
3d0ea65f48cc9d90f32a8d578e3fd38efc0a57d1
6360 F20110218_AACNPU franco_c_Page_088thm.jpg
75a460ec93eaf87b2305a4049f82d688
143a83dd9643379412d399399b9ea2de2158f13f
F20110218_AACMMS franco_c_Page_013.tif
97609d3e7d4a89ee487246c3d6eac9dd
94251f60c23e92d1db1947f44b40305fd2ef2f4a
F20110218_AACMNH franco_c_Page_031.tif
a4b975262be834e9274d11acf44c3cad
a59bc9fec3e344b71d96a28bad0d52e50dd9b80c
6614 F20110218_AACNQK franco_c_Page_111thm.jpg
6efdfda6df170e732f6223d946f9afc4
a290dc2527e4339963ccc41d0033fdcc0b6f63ff
5904 F20110218_AACNPV franco_c_Page_089thm.jpg
41509210b6e608c26d36a4b7af681cd1
95f24eebd1a2bc1568add17c6cfb1c341bdac34d
F20110218_AACMMT franco_c_Page_015.tif
117d5da408a8a32f352792359cd60979
5e11aa472bcd76328ed553660c45f88bad4174a4
F20110218_AACMNI franco_c_Page_032.tif
97ab02c30c0f1e7aa9c4ca8e48b2a764
62890083c3e7fd10977bc006698fde8636eef9ed
6462 F20110218_AACNQL franco_c_Page_112thm.jpg
be58d70d57550fb9221ffa3cec43669c
b3e6731d25943441a3212c4c1e6871e7121dbc62
6040 F20110218_AACNPW franco_c_Page_090thm.jpg
4ca982ca4642a6c2e335614e9dee024d
872f241d0a6af7a60ad6a49671637bca251e65db
F20110218_AACMMU franco_c_Page_016.tif
2b3dc92373b569845ea5cd13bec5fa86
b728a8f7751767675000b9bcfa4048d716c238ee
F20110218_AACMNJ franco_c_Page_033.tif
b5fc16732daf2033889d73da8e977b91
cb96c2c29feef4037ff7782527be593148365632
6487 F20110218_AACNRA franco_c_Page_127thm.jpg
2138944a393be7003704e68db71160f5
c4908dfc464244546d753aa982b07551798a383f
6322 F20110218_AACNQM franco_c_Page_113thm.jpg
ab8f55d935d6f0baeebcb7f7a847c065
8d9525fbc6e50381adb2ea49134953ab51f2ac73
6166 F20110218_AACNPX franco_c_Page_091thm.jpg
a04072a07368d708670ed91803294e79
845891e36b2a52343f5a4185afc524983242df2f
F20110218_AACMMV franco_c_Page_017.tif
d43b3ed83a65d567fa22807643a70214
ba0fe889b3ff01747890d533f803d755ccd0f7bb
F20110218_AACMNK franco_c_Page_034.tif
4572b904a1b269b267943890ad1e5ef0
ad59615eba10c948175a81ed5b1b23b04f2f92ea
6506 F20110218_AACNRB franco_c_Page_128thm.jpg
f7b1bfafb793a9c6c77f12a6bdabf77b
004419c9dc540343b809951da6609dd5c8e370f7
6359 F20110218_AACNQN franco_c_Page_114thm.jpg
8931959da508b7d7e437d2ae6c185cd7
d1aa9ee8eaefbb82209a31199f5672605a87786a
4324 F20110218_AACNPY franco_c_Page_093thm.jpg
f39f0bdde9d4227bc148861aacdf6c78
9ff44bbc34c2d39e6498813e5d0fc18060a62b2f
F20110218_AACMMW franco_c_Page_018.tif
71e7ef981d3c3914aa4e8f4bb1ce8b2d
167cf3fb387446db28a25a1d59bb29a452ead8df
F20110218_AACMNL franco_c_Page_035.tif
b9b177ef426c36c23e575a039e02dbee
5501bb60ce24334df1444f42d3f4b83089e8e283
6530 F20110218_AACNRC franco_c_Page_129thm.jpg
433d381f66056db1fd19dec449d84e6a
c44db2821c7b08a1080f9ce857afb6e306e06e70
6349 F20110218_AACNQO franco_c_Page_115thm.jpg
0596f8575b08ee0805898214062fa20e
eacd6a1e294b4af12335779b61622af287b0b971
5790 F20110218_AACNPZ franco_c_Page_094thm.jpg
bd1e25ad46d8a72c1a3d9bffcd767161
78b52fa8a23765aa3ded74b67c3e8759a7c8655d
F20110218_AACMMX franco_c_Page_019.tif
8a565cd25e3974644059583febad9e05
9b2e7376db5586ced11f10510e62071992642b8e
F20110218_AACMOA franco_c_Page_051.tif
b43213d3ff1f398317e5ff6b2eac6044
b9f7ef7f2d0113bf061a7411c7ed1bc9dc7d60fa
F20110218_AACMNM franco_c_Page_036.tif
f60b72344a25dae8a8113e98004a7a90
23d080fb8f8e0e7554afda6da0046bbfbfe19ab8
6540 F20110218_AACNRD franco_c_Page_130thm.jpg
d1891858c5a99ab66b839a94dad3c6ba
1187665d542102e559ffa13b92b256dfaa8ad1f3
6638 F20110218_AACNQP franco_c_Page_116thm.jpg
c8abec378778313a571c1def0e2137b2
ae9777a3c154194da3fd2e3a8497793e0c2c861c
F20110218_AACMMY franco_c_Page_020.tif
19f168cb512f39766bfc9e5264352551
31b5db8694987c7df3096813ce8afd8c800142e6
F20110218_AACMOB franco_c_Page_053.tif
e401003a2205a0fa2aaac5f52f77d9f4
356c3c13e78a8f698682c108e4c52854b7f57612
F20110218_AACMNN franco_c_Page_037.tif
5a125db0de209d0243f1bc7c7359ccdf
bede4f37f82a9cdb51ef599c6bde4606f77ad975
6738 F20110218_AACNRE franco_c_Page_131thm.jpg
4882c9cd4232747302af4be71eac2a6b
456dede3fd7dc280aa97cc84632d198e8867561b
6741 F20110218_AACNQQ franco_c_Page_117thm.jpg
23e2e158a3051535fe0472b5e8f2b95b
ab389e6c239687afe25c422946139cff33359dea
F20110218_AACMMZ franco_c_Page_021.tif
e79dd7af5ff269f0eeadb9a0ff077725
1ceae4799f4e490066a88c67e21bddd39462ffcb
F20110218_AACMOC franco_c_Page_055.tif
d10c7a128ff0e9443a5a0ded8f16b9fa
15388dfdaf7f3a5e063ca0dad32b2dc573fa3e1c
F20110218_AACMNO franco_c_Page_038.tif
eaa8bf45175acb8d82009988ca3af4f0
b6ddd1d0d0f3d7063b95cac2795a36e6ccf40107
6821 F20110218_AACNRF franco_c_Page_132thm.jpg
57abcdce139e8ca346fa0c537e786ca4
eeacff58cc82a6b6fde86ea12ff9098df17177da
6635 F20110218_AACNQR franco_c_Page_118thm.jpg
8e40f96727b43b52bc09434601e21435
5eeebc4de7dbb09510c574e4bc6ea334801819a8
F20110218_AACMOD franco_c_Page_056.tif
e54af91e4f20ddda913eea85d8d1fddc
cffa9050a05848a691e6834d01782064e5a66315
F20110218_AACMNP franco_c_Page_039.tif
b80ce995eed1823dd89789d7d17ab3fa
5f7934d35f9cfffe3119c3f18b1a453d28f6202b
7054 F20110218_AACNRG franco_c_Page_133thm.jpg
3f05d09d3514f81c31f0b6530f719d7d
581a39a9dfb6ba1acdf278c7d4d62ab1563061c3
6511 F20110218_AACNQS franco_c_Page_119thm.jpg
58b1991111adc025f5bf4993d72afc55
3e8b5052de766c9d57a56f1ac2e9e5ec5d88f5d5
F20110218_AACMOE franco_c_Page_057.tif
22df2870d06451e6d243a182cc148a9d
88c4c33b497e670a86a7c6406f08db14ada25f0e
F20110218_AACMNQ franco_c_Page_040.tif
e5dbda7e20e5a3554542e05067f334aa
3b5c42a8556480e46eae0053d37c4bf050c0f0a2
5473 F20110218_AACNRH franco_c_Page_134thm.jpg
79d1e2c693d4f54fca6b5ff9d97c734c
2e028ec78e30738e68baac2993ae8ecad69b537b
F20110218_AACMOF franco_c_Page_058.tif
67098e71684540ffdd5d99bfe7ba906b
3471337c623d4602d3f5ff7d9a8227cb855d66d9
4144 F20110218_AACNRI franco_c_Page_135thm.jpg
f716df4183a697a5b25da9462277d3d7
779ac9b5824d19a1d6db5c4f6209baddc1812f8b
6737 F20110218_AACNQT franco_c_Page_120thm.jpg
b19f5c51c27aa512792fb3d5d1573d75
55b35e042f0d0f214f4740621ddf478c97da15d9
F20110218_AACMOG franco_c_Page_059.tif
6f4ae225176ecfd2b6678cfbe07597c8
5d5134557ce7c0d209cfdf32a1ce74fc4ec03bb5
F20110218_AACMNR franco_c_Page_041.tif
4ba9c116ce92cc1bd915c72be523a57f
35c8a8c15711a05d9103e889294614ac128b20a1
159427 F20110218_AACNRJ UFE0013409_00001.mets FULL
43c821ec6e8f457589647f871baf3a9d
45ba2ccc245cabb575905a49123d587773dbebc9
6729 F20110218_AACNQU franco_c_Page_121thm.jpg
c72b55907ae58ab05424a1685b01bff3
3e6b58143a1b19f838849e9b9ae4df2f83bae5c4
F20110218_AACMOH franco_c_Page_060.tif
32c78667fc6d82723e37748147eda7be
ac4374039ee92958f4afb906f4886f0e05323a87
F20110218_AACMNS franco_c_Page_042.tif
049975492d3fa60508b5cea123314e4c
d225c3f948ad95ab5bba256bbd03ddb151afc165
6642 F20110218_AACNQV franco_c_Page_122thm.jpg
1b9e6d1f02f302c0106a3b4493e47652
962078500a1b223bee6dddda3041941eeef113bc
F20110218_AACMOI franco_c_Page_061.tif
0760f18e408d363ff986f3238922dacd
ae93f9d0b09d38c559b0a0a9ab6140f4fba39f7e
F20110218_AACMNT franco_c_Page_044.tif
29d5dcdd339f1d1bdb7440451d605c8a
b3b216eda4d29f59ec982daf4511524d293c5ade
6658 F20110218_AACNQW franco_c_Page_123thm.jpg
9c4a2301a53af3e5c2c3b3c516d27fad
265b150f47ec43d14f7b3c140947e603268b0a80
F20110218_AACMOJ franco_c_Page_062.tif
06b5b112cddfb4c54ba86a18bebdfd59
d160c22ecdfcb64cc98b24da33c5500c77d1e6e0
F20110218_AACMNU franco_c_Page_045.tif
611077496d7af9b2fc4756b33a99153a
b5b3439a7395d97786848c8b8f1179d4d3cd1dd6
6585 F20110218_AACNQX franco_c_Page_124thm.jpg
f271e957866e04eb10ac8ffbc50b54cf
8a73fdec240fb727709d1ca23d1dbd46a1b0f1b0
F20110218_AACMOK franco_c_Page_063.tif
a0656a5622b2730dd5fb24110d87a282
844989dc94edd934956cc5ea08c6aa0fe55a01bf
F20110218_AACMNV franco_c_Page_046.tif
b5c8c66ff50a5cce0a85f5c404550768
56b5bacbbe76c770f65426ef8ce2917d456d4efa
6491 F20110218_AACNQY franco_c_Page_125thm.jpg
888da15134e27132ebf71ef90b33c384
65a8c95ac796fe3fede7db5219c92057945dcc03
F20110218_AACMOL franco_c_Page_065.tif
553a6e2e9c18674fee53770c5f846a35
f48de4539fe3b827d783f882545dbff809dc4a05
F20110218_AACMNW franco_c_Page_047.tif
9c046d6d6abcf445850bb5de5220878d
935427938f5866465943e321f15bb5939c053bbe
6460 F20110218_AACNQZ franco_c_Page_126thm.jpg
1c5191adb3340673d51c90f85a226b9f
536537da40e54435d7fa06a97782ec2d9c6e792c
F20110218_AACMPA franco_c_Page_084.tif
fc8b562269a21665769098e21f596388
22e37cf74757be44e72a6265a8a484bad42c64b3
F20110218_AACMOM franco_c_Page_066.tif
a24d7053f2a5b5afa5dc0aafff4ac8c3
5564062273c59aca0fa2c2c546f9baae7dd56a15
F20110218_AACMNX franco_c_Page_048.tif
0e392bc2f99ee9059fe87103e992dea0
5108d0062b12ce664fe2f38478cd20df2ec6caa5
F20110218_AACMPB franco_c_Page_085.tif
aa07ff197b62b2c6e69321b515b1a740
bbd910a614b76f5373781c52aa54462f4bf75726
F20110218_AACMON franco_c_Page_067.tif
4e0a9ab81e70c06898ccaa84c15ff246
1716a939e45d3b895e64ad0268b314a63899f30d
F20110218_AACMNY franco_c_Page_049.tif
7c134dfe7ef645fdebc2dd017b09ad6f
56426109fce42ed1d4850feb3889891bbc83ce8f
F20110218_AACMPC franco_c_Page_086.tif
9fe6bf0e87b37b43c78626a1f5a7cd66
0bb84f25f79f2cc8fc203812f774aa1154a80a50
F20110218_AACMOO franco_c_Page_068.tif
a9281bf79a06ad35b24fd5005d608214
af06a86d675ec61c149a84c6eb313bcef427f580
F20110218_AACMNZ franco_c_Page_050.tif
9b9b818a2d9302d617106c775ec4aa6b
175f08f94a7671b0642fe020915502305c3f3465
F20110218_AACMPD franco_c_Page_087.tif
c12a85f8fd8c3ee8adabd8c79c538fce
6750a10e52348b19d99923d4ab796260c18984f3
F20110218_AACMOP franco_c_Page_069.tif
668b39d3231c648081b2e4e3ecf02cfa
89d3c6f0ce0f25cf78acff7f24072bea1775c73c
F20110218_AACMPE franco_c_Page_088.tif
82cae2f8d1b754c537ca5efb00b45882
3867707c1114f1c66106f57054d07684f261a5a9
F20110218_AACMOQ franco_c_Page_070.tif
94613f8b7ab8fa0b881d3c0e121fc59c
fbdfaf8a44075a2e96c8f630d5a1c320ac87b1fc
F20110218_AACMPF franco_c_Page_089.tif
ef9140e7f10eb78b55f78391d5a2ef4b
5ea5bfd3e4616c3663a14004db7bf4f4a259e7d9
F20110218_AACMOR franco_c_Page_071.tif
4ce9ab07472d5b0eee9229b3aafecee8
11a1d5aa71474be2f9370af8aca257aefa4ac15c
F20110218_AACMPG franco_c_Page_090.tif
848c7e5104b087621604856284eb2665
8d8fd33ae0bca40c4c6da333aa586ec7faa85873
F20110218_AACMOS franco_c_Page_072.tif
bdff2ae06a6d07459e4900ac78bfecde
83d5b00c4abd8c5be8658cfd6ef348e025f22b4f
F20110218_AACMPH franco_c_Page_091.tif
25036cfa1cf4635ddbfed16e94607928
a9f5dd1b309415b3fe6a63b5980ccddfb9815cfc
F20110218_AACMOT franco_c_Page_073.tif
7bfcf41e9af1553c3dd35cef4cda6c9f
0f3da3905fcc1b17ce1ded46c847c9b58612a37e
F20110218_AACMPI franco_c_Page_092.tif
a482b72eeed9558e0178df4fd1c5caf8
c8d467a857a888ad34fca3202429acefcaf8c755
F20110218_AACMOU franco_c_Page_075.tif
60e143014794841473cb545729671f9d
2389f4bfa31bdaedafc1f9e268bef2b596dddb74
F20110218_AACMPJ franco_c_Page_093.tif
5e82a39abdd1abc9c468b532c411443e
3a6dd0b741efaf3b1d53185186770e6016bff5c8
F20110218_AACMOV franco_c_Page_077.tif
f7f031ff9dc0f6922ea50da078821861
4a3726f9d566c35e3350d054c406e0da60ee5a09
F20110218_AACMPK franco_c_Page_095.tif
569310f2fd9ca58577cb3279c4e39d6f
6849d685ee4ac2abf054bcc418462b5f8d4c9903
F20110218_AACMOW franco_c_Page_078.tif
eb848ea2e77c784f18e9d5547ae509be
e45daf8c97b6fd5976410f6cfd0238ab4719ec15
F20110218_AACMPL franco_c_Page_096.tif
ee9e5db54d2515ec6e0b0549e05c8141
2f392f80076ae0ca1a0b7ac296a26fe3fb9b9a34
8425398 F20110218_AACMOX franco_c_Page_080.tif
60d5bee1af226657bd277d04b1883777
b249b38a1d4cac29d3a3730513dead7263edbd93
F20110218_AACMQA franco_c_Page_113.tif
f99f2b421741d2dec593f98c12432c0a
c78d9cb5947ab95e86789cdbc1e2d704dd78d173
F20110218_AACMPM franco_c_Page_098.tif
1b98b62e9db7475ace9badb2d7f19b13
5f7e6254a1d9faf38370f361347c630a51a83ea3
F20110218_AACMOY franco_c_Page_081.tif
2631cec36c058dd14f69088b5818f5a5
ab27c339e5eaa24c558bc2b2f31ce7fa928a4431
F20110218_AACMQB franco_c_Page_114.tif
fc5bc0293f349219e65c349017e1cb78
ea4a3f2a45501754aa89596d9550b06fb40f2b7e
F20110218_AACMPN franco_c_Page_099.tif
43ab2eeea4168c3d566d670fdd0d53bf
69256c2f7f3bf38be7428a5751831328d6164817
F20110218_AACMOZ franco_c_Page_082.tif
ee4d6fe278c0918c7ecbf9828ff35d1b
3ff7584a91c0ba9591265ce754b4c86c736176ae
F20110218_AACMQC franco_c_Page_115.tif
40580d5b0c984461646444cba51194fe
1ce55de82160982abbeaeda73975dce2e9ccf71c
F20110218_AACMPO franco_c_Page_100.tif
bdf249fd6bcd8418fa1df72544ae77d5
48eb15d716d7dacdef37f49585d3e92ae7beb830
F20110218_AACMQD franco_c_Page_116.tif
48e5d08fd74e105c2b3b16d8563bfd61
caa6a99db655f4649c63ca2b6d91c682a4c6d4aa
F20110218_AACMPP franco_c_Page_101.tif
b47541b6b061043a8f4b41c9cf01dcda
f7d221ac13bc01461931eb545d79a84c207c7bd5
F20110218_AACMQE franco_c_Page_117.tif
9ea183a1e0d6fc862e962e64df190feb
cd11e9cefa83b79819f65016309928711d204b15
F20110218_AACMPQ franco_c_Page_102.tif
397c105a99eebab381de419ee158c1be
ef30969a35fc5c508ab15302965d07b7fd4b48fd
F20110218_AACMQF franco_c_Page_118.tif
f7bf559a0a3cfb8da7fec9c463ac2c0a
90aba40e89416b1b122221b4d61f85c0ff68c0da
F20110218_AACMPR franco_c_Page_103.tif
2dfecae4757424d78287f097f9cbca30
c2b590d16b10828c00e1ba28a602d7489cd44bfa
F20110218_AACMQG franco_c_Page_119.tif
dbedb75b295044a1a4a7f033e9a79cc0
ce665a2647b119e9f165406674a99a9322ff326c
F20110218_AACMPS franco_c_Page_104.tif
6fbf0be06e281b4a827a7a1fe78db8e1
e1347f7d7dd17e87d4047e50141afa406148c528
F20110218_AACMQH franco_c_Page_120.tif
1adfb57e1fd3a38ab318ed05e17ee84a
344a6d3a39de85fc5f3416279759049d53279ba2
F20110218_AACMQI franco_c_Page_122.tif
c90ee04179aca26e98eb3b41db686a09
7339d5776d973ae288b51f458bfb8112bb77899f
F20110218_AACMPT franco_c_Page_105.tif
bb3310957d719829857427dbc56d3f97
b0330db60a5d3f4bb61a0df977da0247fbabdfd5
F20110218_AACMQJ franco_c_Page_123.tif
d99aeb6952005d160ae0aa49624f79a4
21a6fb43b0010e0b18e7bf4bf2c693f0296a8454
F20110218_AACMPU franco_c_Page_106.tif
71a4d0fc04fd8e429e90c40f2baa12c1
546f3e4db41c7fb84de9924843c78b4cd3a3d414
F20110218_AACMQK franco_c_Page_124.tif
78121dbe0ad7de5d25e178389f04bd06
7a3855e74164eac3ca0f189fd542c94ee3855c5c
F20110218_AACMPV franco_c_Page_108.tif
5bfacabac182df66531314c6f1080c0e
d6d239cec3bfbcae414d2cda3a56b050f1df7b4e
F20110218_AACMQL franco_c_Page_125.tif
d8a193fde5f8082ccbae2b91a7666ded
bfecf9e5f75196e91230d42f6a87e6ec4be097ed
F20110218_AACMPW franco_c_Page_109.tif
a24a8efca3ee8d0f231ad6169958072f
0b1f06f514591ffe6b889645104365e1f9a2631b
1821 F20110218_AACMRA franco_c_Page_005.txt
557ff6fd939e17ac0dca9834dbc8cc8d
aa32fcb84ad1634e754b62a628e1b9dc9beddc51
F20110218_AACMQM franco_c_Page_126.tif
5b3c4012f4a1c46e3bb8b62652980a61
718e9a6b147fd7c3bf6e4b772eccddc30daecf27
F20110218_AACMPX franco_c_Page_110.tif
88d30246b7a299140046094d589c98f5
16f6fb47cf119d738d678a114cd0f6ffab75d48d
3034 F20110218_AACMRB franco_c_Page_006.txt
3434baf848288581ecb61dfd4675f914
6613cae1efd203d25701f4a2bfe9f27ec54ee79b
F20110218_AACMQN franco_c_Page_127.tif
56e2e18645565135e2fbaa61b5b7cb6e
4f9f15b538661774f8a9db6be7d92fd40fff4b52
F20110218_AACMPY franco_c_Page_111.tif
16a6259f8f737665dba09b7e8decb84a
f61d9a2c8bae6ea0da1651b6f8ed59f72b578cbb
3630 F20110218_AACMRC franco_c_Page_007.txt
dd1bab0a26a38e8864c73dc89f3eda8d
c6d74d30f8d230d4c47a89810cb24eb83e3a86a9
F20110218_AACMQO franco_c_Page_128.tif
59517fbb6173ebdefbdfe8c3e8f4a943
159f47b2eae6240f06f2b138748601769af086ef
F20110218_AACMPZ franco_c_Page_112.tif
896c28ba6a050119bcffe49ee2943a1a
55d81d85c3bfde9c138e9f37eeabd069b118dedb
1983 F20110218_AACMRD franco_c_Page_008.txt
9dca268b02043bf781f6dc223ad81036
09addf64f67223c60f8d0ffef311c17a9a30ab4b
F20110218_AACMQP franco_c_Page_129.tif
6b12a9b6a775d9a28c501fd02d4d1878
f694c4f7dbcba894aab0ba2fb336795f3cdd797f
2511 F20110218_AACMRE franco_c_Page_009.txt
2e378b0e99406f857010e57b00575986
441dd4e8c834cab7f2071f184c778ec2f94577aa
F20110218_AACMQQ franco_c_Page_130.tif
29181277323970f5c21a13c5275a608c
36a47cbe2d83746b9cc910ad1a1e96298c306040
835 F20110218_AACMRF franco_c_Page_010.txt
de19e1360dd0c3d8a736ad4df2081ba7
ca35bb030fc6aa9d18a8ee1138aabd062d911f66
F20110218_AACMQR franco_c_Page_131.tif
88d9d1f0b4d1b11eedf67b82b630a46d
80985b34276b065c37f105380f0cc1087eef9147
1551 F20110218_AACMRG franco_c_Page_011.txt
648d50087c89d6e8fabc3741e4b53ed4
b28e4e24853659b5056c1a00b1cf2a8b9baa2ab0
F20110218_AACMQS franco_c_Page_132.tif
4b0f730f48858960b112195486528380
501eb99c2325774238580d5af2f85433136aab4d
1616 F20110218_AACMRH franco_c_Page_013.txt
86fb477a8811d358dadda02da5ccbfc6
60a16cda560cf954e0e8ccefd3deaa5291c7e85f
F20110218_AACMQT franco_c_Page_133.tif
cb658a4b396ff539f4e8462b42ea9b6a
1b50df99dcf6c3b9836f296af9c8d5964b3d7dbb
1927 F20110218_AACMRI franco_c_Page_014.txt
4943a0fca482f59c9d717184a7692573
82c08606256ab91126de1492df940c9993004404
1929 F20110218_AACMRJ franco_c_Page_015.txt
288aa0415d0640d21d97ef79afcbf28f
1b1fdcdf5d136aed4d1fd83e2c9ba6bd2512b42d
F20110218_AACMQU franco_c_Page_134.tif
1dee883f87e0df505c0928f96d70689c
055aa6a24bda993ef03d0c51207efcc728e2e8af
1805 F20110218_AACMRK franco_c_Page_016.txt
2c11b3cd6196f2ff4562e62008bc179c
4ea46095422d42563f58fb81fa894d4d01e5e468
F20110218_AACMQV franco_c_Page_135.tif
df4d54c080c47d104472e428f172f342
9fb99dcc2c8fef5957723531eb3e9094717f5df6
1816 F20110218_AACMRL franco_c_Page_017.txt
f1453be53aad9ccc66004557a142a91e
3af58738d1fc121498babfcf04e7bfbf4c567795
517 F20110218_AACMQW franco_c_Page_001.txt
4831125d057524565192fbcdee2178c1
2c71218869c137fe89e83d0e6b691d294072c8af
1809 F20110218_AACMRM franco_c_Page_018.txt
047730413936af6b253dce9ef51a52d0
819a86c5e4fb24cd4d977f3db23eda0571412509
113 F20110218_AACMQX franco_c_Page_002.txt
d12bdbed7c8e5f75c25ad541847168c0
1a9dede3de1a72bf8e9511d370f9aaa9c4ff011c
1973 F20110218_AACMSA franco_c_Page_033.txt
5635585abfec4c5b2771854b281c688a
b5b9e8ce2d44ed940285bf47651ff0ffa045cce6
1981 F20110218_AACMRN franco_c_Page_019.txt
d2734ba3d3cc5da451a9f46810fd9249
2ae1f130a8e490e4122fef97aa6cfb0ef3ead84e
682 F20110218_AACMQY franco_c_Page_003.txt
8a6bb6bf0a8cc6f2e07200a97b555c92
83a13dabce676de3452e6d928bf8d31dd2c0eaea
1845 F20110218_AACMSB franco_c_Page_034.txt
1cad70e72c2c42abd4f0ccbea85f3122
df5376b7673fd53939e3de3c1e2212398dcf3e1a
1975 F20110218_AACMRO franco_c_Page_020.txt
e2787a91686ba46ff327792ecdf3ac4c
1ddc882fedd0d3f73451af6db7ab7a6ac144d84e
1704 F20110218_AACMQZ franco_c_Page_004.txt
4b99c3177d3fa37b7054f83e06bbf486
15c8211f9a28de330290836735d0079e60557ad6
1968 F20110218_AACMSC franco_c_Page_035.txt
deec14e837f1f71024a228c892ee7d2a
2692711079e3d3d735026b0a7f9f667ca3432be8
1913 F20110218_AACMRP franco_c_Page_021.txt
d74a8545cc20eeb60593673da2eaaf95
3b39343cec2072a0990f2735ad8d11c0bf545580
2026 F20110218_AACMSD franco_c_Page_036.txt
23bd8b062087cc62be8e5a2194d7710c
3af3243b13859afaa3f6f4a5c7ac175cd12711fa
1868 F20110218_AACMRQ franco_c_Page_022.txt
299e0e1bf1d0a5173c2b2372e4bb189f
42c89128c76d8d6459e542c7d949b49d063086ed
1993 F20110218_AACMSE franco_c_Page_037.txt
c80be5f1af4eb5816e95e1df7176270f
59a4859e9fd83f8d3e1438dcbc5af4c0b04d0a7e
F20110218_AACMRR franco_c_Page_024.txt
865da3d3b5800acb980650961597c833
1a32730cf0a18c1628b99a46eaebf9b87dab7d68
1966 F20110218_AACMSF franco_c_Page_038.txt
7f78133ac13b6ee00f37895c0c355f31
edd5dbe33ce24e4dae9dfdb0d5574b65fbd9e45c
2081 F20110218_AACMRS franco_c_Page_025.txt
0f85bca092bcebf62137c7009e2456d5
f82ea848f516892ea1437c7bddef9760a8e300b0
2039 F20110218_AACMSG franco_c_Page_041.txt
b07e738a32a9e8ac9b86584f1dc44e89
10b996804e8cdf1bfadac5a15644477f0761d1a0
2106 F20110218_AACMRT franco_c_Page_026.txt
393ef8ee6b4db23ace20564d5f3f02bb
389b7cd4d8e03a00597c28507674d0ff2c10f58f
F20110218_AACMSH franco_c_Page_042.txt
5a7b9a1c4b564d562b9a5e8031aa60e5
0b02ab301ef7f85eb99e9fc7a0bec11034e03ec2
2035 F20110218_AACMRU franco_c_Page_027.txt
e34242ab522b0632dcb91a83bda0f94b
6055d75132c6f06b6fd19f689e921f1164bfb0ba
2082 F20110218_AACMSI franco_c_Page_043.txt
5861dc64d928ea366e3448d7e990630f
7f141a3d46938690142f761eab6c358eb585b32c
1016 F20110218_AACMSJ franco_c_Page_044.txt
e04474a571e489d839b4e52e9e21e7bf
045da1ef66e7c9e3737c7ad80997d043c7fa2f43
1881 F20110218_AACMRV franco_c_Page_028.txt
6ecb2c929c8d76f7ccd05b56980f04f9
9e2039ccd198692df9c99e5a113ef1db7f44bca5
767 F20110218_AACMSK franco_c_Page_045.txt
94b7728f8db1e29df9afab922fe855ce
c1c533d2efafaa358c89b8d5d7de4db94a13ab4a
2053 F20110218_AACMRW franco_c_Page_029.txt
68e48130901082e33cf0d46365052316
f4fa44ddac1577a10dd60a96275bfa4d1121e73a
1806 F20110218_AACMSL franco_c_Page_046.txt
d2b4fe54a60dbc5d4c598e1cbd954f0e
85f0cab4f87d2fa0dcd5afe1f8985265facf7454
1937 F20110218_AACMRX franco_c_Page_030.txt
7c4d7f167050d875d61dd011c5769c8a
eef6bd325dfec1dff36ad5f9a628d9ed0daab684
1908 F20110218_AACMTA franco_c_Page_064.txt
c296befbb584d9be3c32fa4edc486f0c
33b6d9d92c7881a2526cc9d9dfe2cf103e145b72
2078 F20110218_AACMSM franco_c_Page_047.txt
daccbffaeeff4df76695096788efe522
f954a6027622968bef6b3fe0c2672112a912b4bc
2085 F20110218_AACMRY franco_c_Page_031.txt
2f30e56ad9e660a647bb970ac5a7c8cd
8bf271e039bb5142ae7ab179c152f698212ee3d9
2016 F20110218_AACMTB franco_c_Page_065.txt
7097612169c81c9b397526c16bb55dbc
815262e1236e134096b75f69dd2edb397e551d19
F20110218_AACMSN franco_c_Page_048.txt
3dff210ec403dd1b61d1286f549649e7
0a5352fbafa00ca23534d6a8012b0efbf129b726
F20110218_AACMRZ franco_c_Page_032.txt
20e73d4ae1a1035edcab3472198ee54e
4c1786fcc67c9e8bb6e5ec03f1fe9977a0930ac1
F20110218_AACMTC franco_c_Page_067.txt
a31c4d3ba82124caf37ee890cd59bcf0
f2fa7534cdaef33729641605f55bb2d19353fb8d
2006 F20110218_AACMSO franco_c_Page_050.txt
47f9bcfe78f331da38ee588c8e20b6d3
f4ec3b0b033ab1b0731e06e11d64b1c5d4ccbe4a
1990 F20110218_AACMTD franco_c_Page_069.txt
99e729244ba2fb112f29a0e8a88a594e
78dc17a2f97010800f58cb5ab61cccc55904f3b4
1999 F20110218_AACMSP franco_c_Page_051.txt
101f1f26700d5fc4eb589c67c46bcb6f
def3d49dbe743b1b64f74bdd2343b67598a28da0
F20110218_AACMTE franco_c_Page_071.txt
1e36c8eb1501bc265054a59fce563839
4e23fa2cfeaeae98b9f0c79fe9de6a5099cc0d71
1941 F20110218_AACMSQ franco_c_Page_052.txt
99671b67caaa18029e9c2ab12dc9269d
cd0762c9f1925d41b15f3a0016db850049345ab9
1750 F20110218_AACMTF franco_c_Page_072.txt
ea47d7ad8e497a3b08db13d847d838ec
0ec9e45215fdce02d6ddc7c84832671ac0958eb1
1985 F20110218_AACMSR franco_c_Page_053.txt
47e7cea89d5a50e2a35a320ea6d9161c
fd856e64fd40683de55ca4df1e5e1119f1046bc3
2030 F20110218_AACMTG franco_c_Page_073.txt
81543d35f521138a61c696507fd35290
ce08ae7bf49a48a5aaae860c1dbc4cccb004b927
1872 F20110218_AACMSS franco_c_Page_054.txt
6da730428e9e771009cc8d3297f9f12f
ff260c30eb653c72c45a6d0e181c7778e078245f
2041 F20110218_AACMTH franco_c_Page_074.txt
ce7f52b0734d68c322afbd3839a11872
047e12cf40045d039eaa89d777131f297ff2af39
2108 F20110218_AACMST franco_c_Page_055.txt
0b7d79f8a1d4aa110a2876d34548bf11
3d8704b9d839f2787b503b4d1140cdc0742438c0
2089 F20110218_AACMTI franco_c_Page_076.txt
2a7df15453e060d488bdac1cb90d421f
9a7cd8497aad6a09f55b85425e4015e938fbf67c
1900 F20110218_AACMSU franco_c_Page_056.txt
f249510607462e4cdbfd1b8b1e242e8f
753a4b89f76cf08d977e499a3e15e5f002e99b60
F20110218_AACMTJ franco_c_Page_077.txt
181daad9da6552701bf7c11ca9be86d5
5c3f73f1c044f1b455d32237d9c385f07dec63f7
F20110218_AACMSV franco_c_Page_057.txt
2de3450535fab3506d0c9032343ef530
dbc676a841c31745613049465883cb111aa823e2
2062 F20110218_AACMTK franco_c_Page_078.txt
47a7d5f08d6c070b6386ebf2e980f2ca
2518e2b31a715cc377f4c881bd0c87f2f208a09f
218 F20110218_AACMTL franco_c_Page_079.txt
e29398fc096a13f652a0603ec3e4e3f7
d98d26bfb498b4ccdc512c3a6dc075edaf4a2f64
1896 F20110218_AACMSW franco_c_Page_059.txt
3c180ebbf8aa213fa5a08823b703217d
46a5a358d6fd945c769ad14d5c933959010e8411
2061 F20110218_AACMUA franco_c_Page_095.txt
c3cb96561f57cf5b0e13a0843a335aa0
28712d5a08655afe75af786ae14cab57d2b638cd
2219 F20110218_AACMTM franco_c_Page_080.txt
7edd2b531042a218c29999b717037128
25a82598e3d4750606afe7f867e78f2ffc1cc0ff
F20110218_AACMSX franco_c_Page_060.txt
6c68df1da536edd57fa5261ec5b4bd22
e28469c078a5b54463c849b5550fce803def3526
1918 F20110218_AACMUB franco_c_Page_096.txt
46f705c619274a78ffc7f0d1a9af1a45
5a68ee456238e14eb5aeb65ca76957fdcf979860
1309 F20110218_AACMTN franco_c_Page_081.txt
db68b5f2bc31672f66f98f868c11a3e3
d1b0cb39c7261624b069abea9918962b565ae984
1556 F20110218_AACMSY franco_c_Page_061.txt
26fe79a1d8f8b947ca67ac7b79b3cc6b
00c682bf35d661c06805784a9864d3480ada7759
1914 F20110218_AACMUC franco_c_Page_098.txt
eb4e9a5c3b708c06291526ffe9ddf85f
49dd388dc5a6c348d4992f0f85c68f7e303426be
1605 F20110218_AACMTO franco_c_Page_082.txt
3977ea4ea5fdb9a04b98d64039162baf
af8151a5eccfc7bbc5fb46628dc4b470c3f4b669
931 F20110218_AACMSZ franco_c_Page_063.txt
14667509c73e30763fed5117557dcb2f
8fcd5d421c2cdc8015b80696926bb6394d640da8
F20110218_AACMUD franco_c_Page_099.txt
5e63c606202e0f28e44f970871b43123
28c4547ca04d5fa58cd57653239133ff324bf616
870 F20110218_AACMTP franco_c_Page_083.txt
d3d2347b393088c94d30057c436fda60
f481cad4216e50c71c024190abc1d94545c8fe47
F20110218_AACMTQ franco_c_Page_084.txt
65ac73be7963e0841a2861703aab08ea
89cc8c8630462cd49c02384d58e46c75b985b99b
1955 F20110218_AACMUE franco_c_Page_101.txt
00d3ab97257b8915cbab89351303c927
51677817d3fd0c71953ca550bf5698a1b3533679
2019 F20110218_AACMTR franco_c_Page_085.txt
3f52f293132767e1d5ab1d85dcc7b7fb
46673f7ca3139133aab7b8abcce847afeca4af6a
26711 F20110218_AACNAA franco_c_Page_007.QC.jpg
e7f2427b734bc533527f6e017c4c60a3
7e0dc2ec6082e9862039a81ddaec64ba902466a3
345 F20110218_AACMUF franco_c_Page_102.txt
c3fdee755abab464536a7ebf16e7c6f4
1b46b816c73978afc2fbcbb5d22279ec0aed5dca
1991 F20110218_AACMTS franco_c_Page_086.txt
8fe36d05a261a659b3862886ed92274e
2b80fe9e3a6a33863719a4b1343b8c4011d3d18c
54140 F20110218_AACNAB franco_c_Page_008.jpg
6ea8d12f2ee15385f5b7e1fb099ebe5d
0d9599d198c61472ddc8eb3eb5435456777f5137
2453 F20110218_AACMUG franco_c_Page_103.txt
46fcbfaca0b7678676a20eba887c23e0
a46cdc6de85189d7e5a8e8143d0d1812301e15b1
F20110218_AACMTT franco_c_Page_087.txt
06b253d8d7fa4ea8c5223fca3e7d6736
440527b97462fb1c8726d564efc08c3926774447
12853 F20110218_AACNAC franco_c_Page_008.QC.jpg
287de22de5cac28594d8cec8fca40fdc
57b7d66019b333526fe9cfb0ad8da21d78e3e693
2723 F20110218_AACMUH franco_c_Page_104.txt
916382c97b07ea8a082b04d2a9cd19aa
6ddcb42068f16e211b3c1a9ef3ba6987a3bf58e2
F20110218_AACMTU franco_c_Page_089.txt
4ba25e68f796ec2343bd9e68ab2b771c
7b85742d2191beff56e065f1d5494ce6c2c5b587
2693 F20110218_AACMUI franco_c_Page_105.txt
0dd6e1c408de5bab488ae2be9a695425
966ed57615ae2613b4ede27c292968c312f991f5
2070 F20110218_AACMTV franco_c_Page_090.txt
9db5617a4f9294a9cccd5fe6b4d1442d
756d4915ba00af8749f44903ab84064389fafc4a
80049 F20110218_AACNAD franco_c_Page_009.jpg
086ab3ee57a19ae88ec80698cf208a3a
fbc596a004b1ee08fec88acdc719b50db9bd9157
2361 F20110218_AACMUJ franco_c_Page_107.txt
9abd46fd4c55dc51b66cbe5e51e54bc9
14f93f29fa2c94d8bef118d54c9ee27521b93ff5
F20110218_AACMTW franco_c_Page_091.txt
3357659456eb45a8e9340059bb4ad63e
f2f3554fe0726b8f3cfc6bd8213e501621101871
30659 F20110218_AACNAE franco_c_Page_010.jpg
2e275950ca7776cfe3623192f63ae94b
16560f00bcdd0099cf31b7a5d941d85bbd65615d
2497 F20110218_AACMUK franco_c_Page_108.txt
8ee3b6c22cbfa9c849bf08d294b33077
adbde0f2b04c61e78b7550aa61823991f9a24ce1
7942 F20110218_AACNAF franco_c_Page_010.QC.jpg
a7c56b4c6bae9ef1dacfccdbff7a293c
53951f72e2c1fde438a3cac5aedda42c339a980c
2440 F20110218_AACMUL franco_c_Page_109.txt
0f3c30a3bba733e94687d1e8b771717a
d64ab8ae2926dd8f1cf366a6e6922b67c074fae5
1987 F20110218_AACMTX franco_c_Page_092.txt
ca16d8dc93c241d98d5b6c8e113cb478
a2f8e2c3f2906186d2c156b75a0787ec15a1da00
62011 F20110218_AACNAG franco_c_Page_011.jpg
427c74a395d43f2d1e8d43164cd5bea4
db84f5a7c66cb4c0b5566ddb78d6d232b978be18
F20110218_AACMVA franco_c_Page_129.txt
2e948734f4751d02fe657ad0bffd7ab1
864558c8f3c5a4911ce8c08a1c3cb966cd726fee
2532 F20110218_AACMUM franco_c_Page_110.txt
d6f22d030fb59bedcbac386664f7b9b8
f44d0ca38e6a2907167dfb201b55c11b1ee221fc
1700 F20110218_AACMTY franco_c_Page_093.txt
209b530903ca7693521bdd739e599c85
8ddccdd6484938d5348ddf32827765796c249ed6
17408 F20110218_AACNAH franco_c_Page_011.QC.jpg
c30cce62727641084406b9dea3ad9545
3e1d1df39193ea646bfa0edb490bc05212957e74
2544 F20110218_AACMVB franco_c_Page_131.txt
0334e934559df541b4fce828069e4320
918aaeab6b1f01c2b8c8806b033aaf965118a01a
2457 F20110218_AACMUN franco_c_Page_111.txt
f1876135e2f4dd20080d6891ced027f0
e6bab4089a9f8306c792cbc0a8a4c9092053929d
F20110218_AACMTZ franco_c_Page_094.txt
355092fd2fc46fab65462979384715fe
c6f4d2f1c48734524bf538df52cdb82a7844b994
47914 F20110218_AACNAI franco_c_Page_012.jpg
95295421db3afb42f46fe792cfa47327
5ae453b700f2d8b2ae0f6e4a4a011480a068314f
2572 F20110218_AACMVC franco_c_Page_132.txt
c9e75d7b669219737332b1ac291e06b0
064a799887bc982baf66ec8fdcdb39aa26eab325
2377 F20110218_AACMUO franco_c_Page_112.txt
a287e5e962eb7201f61647ca4166c14a
c337b853d230133ecc82d3aec87266da1f0b957b
14703 F20110218_AACNAJ franco_c_Page_012.QC.jpg
07aed28cdb311494c7eaf2f54def9400
4507a559ccecb284de47178b823fe35cb86abad9
2744 F20110218_AACMVD franco_c_Page_133.txt
8c67cc3037ec543273761bc93a6cf3aa
d98444bc4864809d41d1624ae3e299ea52ad09aa
2423 F20110218_AACMUP franco_c_Page_113.txt
14dcfea55e5991ae967d23d92673cfa5
cb68a5acb4d487c7031e8c133a8d94b5be6ae678
65133 F20110218_AACNAK franco_c_Page_013.jpg
ac00d95a93da63e57537c37700aeaa6b
8c09a975d3c0823e45416ad15d9ae7f52f4451fa
1969 F20110218_AACMVE franco_c_Page_134.txt
35db042343c3ec6ae0e5823ef7150e17
b63c5ab036eed30ee17f90383d1668eb308461b5
2603 F20110218_AACMUQ franco_c_Page_116.txt
037d208a0be8cf8798b32fdfc7dc6803
2d19ffc649e2170f00bbcacd659e236c78b85030
76671 F20110218_AACNBA franco_c_Page_022.jpg
d1a30078f88166d320c0d0c425d361e2
2527b3ad239ca4b765db7c70e176a814564d5e88
20254 F20110218_AACNAL franco_c_Page_013.QC.jpg
a8e0ef3fe835dcf9d6a39a7fcf434df9
1bfbae0cb134bf1b896a41cf4da8142b799b54af
1247 F20110218_AACMVF franco_c_Page_135.txt
8992e3ade87851e8d6ddbc401b26ccc1
84be5b428385ddbdd96c4350cac126ee0260bb11
2621 F20110218_AACMUR franco_c_Page_117.txt
cf2b22ed0b7434537f0f7922e7c36f0f
aec0c8ad03268771d044fd05a81697b383db9017
24475 F20110218_AACNBB franco_c_Page_022.QC.jpg
d60986de02738b7f1eb551fc45d6dd47
7396d606b2252fced537f7a0329c2a52b1c982a2
81374 F20110218_AACNAM franco_c_Page_014.jpg
69b2c07cf38f514c74e91c47055b9086
e5512c8d52216d4b6aa01affc53f065a563aa960
9260 F20110218_AACMVG franco_c_Page_001.pro
0eb5803d716c5c3af28f4dd32f4a1a90
8c03573e3db5de87ee8206a4807bdfd9e8990c77
2515 F20110218_AACMUS franco_c_Page_118.txt
98253aeb79fcd5f731c80530f05963ec
c247e2940d84e60edce5942ef1a20103ddba9cfa
25493 F20110218_AACNAN franco_c_Page_014.QC.jpg
f8ced99169ec296f3162b467cc3a43bb
0a0cd64068aaf277a1b1f9ab217eb2761e44eb7e
1198 F20110218_AACMVH franco_c_Page_002.pro
fd1fe350b2c70e3aa9c08a2f3ad9d6e2
438838e5b503df878437f3c3233308b835e9bc23
2805 F20110218_AACMUT franco_c_Page_120.txt
02e36fba14b0a0ad30cb55b830a401a1
9b2e5c0e372b68fe53499d7fecb0ad75c9cda147
87110 F20110218_AACNBC franco_c_Page_023.jpg
f9829106922fafdbf2a6c7f79e71d025
58a571c1485596fd1ef31a018e2761b5a8d3f897
79093 F20110218_AACNAO franco_c_Page_015.jpg
7792f3085dd7e767f05bb9c88b72e130
b6116a8dc4042b34892c0d07c1558a2c7c8954d3
14070 F20110218_AACMVI franco_c_Page_003.pro
fb37c1f65eed4c3afcd10fc2c1f204f4
e52772bde65c407d8f4264d89ef35ad474964361
2564 F20110218_AACMUU franco_c_Page_121.txt
258a4776135815adbf5506d5fde5e76e
9af992db98748b7d7c64d8f76564c99dcf6d799f
27136 F20110218_AACNBD franco_c_Page_023.QC.jpg
07849249f867e0b2ee1ede8a4b9d23f3
b8ee741caccfbab7be40480829874480884014db
77597 F20110218_AACNAP franco_c_Page_016.jpg
ad98c89c8422c947acbb11aa617f4786
362bc231a4e851ba2905d551686fa577e7986c18
41799 F20110218_AACMVJ franco_c_Page_004.pro
67fd7afc277c1e743cdb38f2f45a86ea
8f178b7f6b9b6b0fd22e44f882bb6765e611fb64
2512 F20110218_AACMUV franco_c_Page_124.txt
d58fc7cf47b80f9c48183cde95884877
0f8b7635dd7cb1ca00027f182b0532e2fa367769
23893 F20110218_AACNAQ franco_c_Page_016.QC.jpg
e81900f85179567bc601271c303906bf
962ecdebd0dbe64b57836a273022b0aec8ea87a1
67061 F20110218_AACMVK franco_c_Page_006.pro
a3819051f474d7e472d77d0ea023ad0d
c725e63dfc02ff47ad0d552a69fc8be8c4d7bc3a
2461 F20110218_AACMUW franco_c_Page_125.txt
4cee86cb5030470c78e8969ec87c3004
2e59924e060eedc205de3ae2d9d720dc598a9d77
81970 F20110218_AACNBE franco_c_Page_024.jpg
f4daa64b4125d915e436fdb306ce1186
a4b9a7c8ea2759e906bf5e5d351343717904528b
76120 F20110218_AACNAR franco_c_Page_017.jpg
167b46ca69ebd279e4d788aee45a4fd8
80457c6076282f6a2f6bc72f95630c81c314b5cc
85392 F20110218_AACMVL franco_c_Page_007.pro
c161195e2e9f6cded0fd8ca562a9ee77
21fdb2c59af59fcb6aaf0faf5722a9465540f9cb
2573 F20110218_AACMUX franco_c_Page_126.txt
78e86e4c8d37f0c69f1d72bcb6bf1edd
b96c31179c6b0952195df19afa1e87fb9c0274ab
26330 F20110218_AACNBF franco_c_Page_024.QC.jpg
f6e7be3c3b56084d42badab98ab335b5
2947d6d11ec9c78d6ee2317a6592e772cde4be0e
46867 F20110218_AACMWA franco_c_Page_022.pro
32f04d4636f6e229307676f4d2531d96
86e1b8701614430aec1d10615fd3936274e027ce
23995 F20110218_AACNAS franco_c_Page_017.QC.jpg
5df26984205d961ad45eda44a6959394
d121c09d6acf9803897a84227b3eebe07da332af
45779 F20110218_AACMVM franco_c_Page_008.pro
be4d7945ebfb2ae405216e05d23cbb98
116ce18681aaf96601205d4ee9cd060d8740aabb
89419 F20110218_AACNBG franco_c_Page_025.jpg
cf7695a76eac65e0f807985289d4a211
6641d59b55dd5fb6d66f720dc6d770842f218322
52107 F20110218_AACMWB franco_c_Page_023.pro
4cac3e2e9c0730ad7fe22a8b7551513a
aa823bd4b8f4a60a7d945f32e22ad550a3609125
76486 F20110218_AACNAT franco_c_Page_018.jpg
fc8785dc501b955414a3e77e15a39b75
91ce1e2af5f42b44a79cb3742509a8117a9b4685
59493 F20110218_AACMVN franco_c_Page_009.pro
a110a296d097e3789825099675026ea1
11d3599c60d4050d4e92d7e33541215bcdd0919b
2502 F20110218_AACMUY franco_c_Page_127.txt
586ead8003fb70c7c824ba9832ea2262
a074c482e7cfc47de1bc235824b74b7e81736d60
28063 F20110218_AACNBH franco_c_Page_025.QC.jpg
2da1852a7e9c4f9e7cfbb35a9054e375
361839f3462cf6964600655ab52e5512d426f016
49722 F20110218_AACMWC franco_c_Page_024.pro
9521faaaad46a5c93f6e714e892c4897
b848596dfc68ee22225c21b26069a164a8617d0f
24013 F20110218_AACNAU franco_c_Page_018.QC.jpg
4863b5b73e39448c0b0bc8dfccc1c278
8442f00bf59d9eda0f6cda74353992343f395f5e
19994 F20110218_AACMVO franco_c_Page_010.pro
658af935531a9191aef30480fc2e9a32
924cf4aa29d15644152250cdf05b5d6158e75afd
2562 F20110218_AACMUZ franco_c_Page_128.txt
11dde194f3e0aa6411f62c10f776a385
a8d3e8e98b04d0aafb473675f901c0f44e7afd01
88882 F20110218_AACNBI franco_c_Page_026.jpg
2676255c814465dcdd2f8d1c89c6e58d
c20c51081dea5875c8e71b601832afed9d0eaf9f
53042 F20110218_AACMWD franco_c_Page_025.pro
60dee5b1389374e3334f7df7ffc35b8e
e80b9fcccf05ef29e5b43fd5af8634a4c405f0d1
82161 F20110218_AACNAV franco_c_Page_019.jpg
1ff3ba6d8b78687fe8c493f295921821
ab8f623b99ad254b74154c58de446606654997f1
33934 F20110218_AACMVP franco_c_Page_011.pro
f96adf8075cc4927edc590a714add2dc
32a06420b05a862e56b1a265e66e886a06bb11af
28323 F20110218_AACNBJ franco_c_Page_026.QC.jpg
74d67d820d52e5a0304e9f5ae2470547
f4c229a37370c709b6d531a7200e10c07c51ddd5
51841 F20110218_AACMWE franco_c_Page_027.pro
450c20c0cfa40507303a55c04f3e781a
69342a883ba2eff5cf3e0fb71994ec64e6bccffe
25676 F20110218_AACNAW franco_c_Page_019.QC.jpg
6ac6f7fbbf117af62ccdb84ba2322519
bc5fc9ebacd14185f445f9185817185af4e75062
27883 F20110218_AACMVQ franco_c_Page_012.pro
26fc74f2e8f49ce879f1bcd677de7ffb
aaf0a50a0e9c4ba33616c1269fd100b5dd4cef0f
85365 F20110218_AACNBK franco_c_Page_027.jpg
4dcf38285aa64b1ac8c1c933be2c205d
e65ac4f63e13a946ce066609335a7b5b9511eb26
47381 F20110218_AACMWF franco_c_Page_028.pro
d4816a3cc99fca698440729fc9176366
be759e2702644113eab37345f42cba1cfe1b53cc
81898 F20110218_AACNAX franco_c_Page_020.jpg
a3eeba1698cec822d06c69f50870eaf6
2a3462621bab96b485d7f014d6258933668d8a94
38047 F20110218_AACMVR franco_c_Page_013.pro
3e40a35bcd8005b6abfe05e3e9c121c8
f9f4385cd6623d769417ea4af2e66bbf86f1d197
26318 F20110218_AACNCA franco_c_Page_036.QC.jpg
a5001b444c3a0bd0dba89eedee46c8d9
94ce45c348605194c7146c377e59339fd009b4f4
26815 F20110218_AACNBL franco_c_Page_027.QC.jpg
13223d4dcec9a1188b70092bb0341829
89fb6dfab31bbc68959834e8bfc3da684eadb17f
51887 F20110218_AACMWG franco_c_Page_029.pro
8517628c9fa6cc6fc93bd07408303e91
9d02f1401c389561b35ee71d368600a6ac545c28
79727 F20110218_AACNAY franco_c_Page_021.jpg
45276c0621d29ddd2221607bd307f8e6
750d3ff0b42e62e675261c3d7a7ae37886b4973e
48390 F20110218_AACMVS franco_c_Page_014.pro
419263d020096d68278a5b01691f524a
e3a9a37094fccaae4b24fb84e733c6bc9d9cab0b
83765 F20110218_AACNCB franco_c_Page_037.jpg
ad30f71c093f4679fd399037e8a732cd
c81b3dc9ecffa475a84e0b4969f19c015818fc08
79762 F20110218_AACNBM franco_c_Page_028.jpg
30f289863ef925c20f069b46a71d6415
0c43c47db9197d3b14ed484f1cae893f79f9b55b
49110 F20110218_AACMWH franco_c_Page_030.pro
418fd80e2168463af281ed9e6ccbb3d8
5643405df9ad8a1ace53882bde507aac513c9b47
24484 F20110218_AACNAZ franco_c_Page_021.QC.jpg
27f225172cd1b8a6f2e66f78951fda11
1ba7a63d39d5ad33b9358f67b3c984f9e3c7dd7b
48701 F20110218_AACMVT franco_c_Page_015.pro
4b18255176e13bf526bdd8f414db90ed
f992ec906b448c269872aaefdba005fd6a5d9186
25497 F20110218_AACNCC franco_c_Page_037.QC.jpg
117c444b45273757cc964735b91e9a43
0ffab5b14051b59956d02b1250b8e7f42283e270
24215 F20110218_AACNBN franco_c_Page_028.QC.jpg
45e75bfd371f419869f5297df12d7f56
1d201868e5560bad58ba643fd50b61684a704947
53041 F20110218_AACMWI franco_c_Page_031.pro
472869c134c2c8aafe1b59ed7c04110a
379a499d8ed905f655f3a183672a50b354b4fac8
45495 F20110218_AACMVU franco_c_Page_016.pro
8fc15eb7ca130c394aaff41b20758bec
e6d2ad55941ccaafcb42e17cfd06a7c40b435b3a
83170 F20110218_AACNCD franco_c_Page_038.jpg
21066641e4b1759ea7854ba2afbb1489
6a52b0dc95528472019154ccbc57e3c349c0cd91
85247 F20110218_AACNBO franco_c_Page_029.jpg
c18a944512c00262b855a0145d8806cc
ef28e2e60b895c8410e10f5a5337ffa3fbac3e4d
51942 F20110218_AACMWJ franco_c_Page_032.pro
7a05290230eeb401774c280a43f21353
643adde6ef80abb0ed930839c087800b9a1d5e4f
45724 F20110218_AACMVV franco_c_Page_017.pro
5c63007e4f5b18573e9a647381ac4ad2
4c5fabebfa1daa3ad24c14ad82706a69be6b95d0
26796 F20110218_AACNCE franco_c_Page_038.QC.jpg
5e7cfcde06edede729ac970b180605ea
6b0360ffd0a6368177dd722a52a570d0babeab04
27409 F20110218_AACNBP franco_c_Page_029.QC.jpg
89dab7e34358f2be42c56c9fab33ecc7
0b16e327b270dab41d9d3a456de8cf326bea0ee0
49947 F20110218_AACMWK franco_c_Page_033.pro
3e0f93f0d5fe7c416a32c4d00589b798
2a87393380e02787ce61d443af394d9eeddbe976
45594 F20110218_AACMVW franco_c_Page_018.pro
3007e782c942c1daccf1010529ee29c7
654f636a249a57cbab5aca3d63c5d4d01f3d0faa
81523 F20110218_AACNBQ franco_c_Page_030.jpg
33913c00be74a7043585b76475028a30
e6b59eb240c536e8a2291b22158de83ef8fdfcb8
46548 F20110218_AACMWL franco_c_Page_034.pro
18c199ac7b4e08ef5a7a942820b25c42
0c28cf9ca16d59e00fa775e222c1ddd0615b2ce2
50010 F20110218_AACMVX franco_c_Page_019.pro
6fdf8d8db5fc4076576f621975f139ac
097759b6646ff99167fd639fba93cabb49afd513
76785 F20110218_AACNCF franco_c_Page_039.jpg
07e9e0975d3659e69cc353832d5476c4
251659ad4db946a5d2e2ad3025de8e6cf1479849
25403 F20110218_AACNBR franco_c_Page_030.QC.jpg
b4a325575eb0292fbf8d593b73b4cb92
3dadf82930f6fe7f09136841076319a1fecf3119
49983 F20110218_AACMWM franco_c_Page_035.pro
e2c1f3540889b3b1f6f364645ddb7496
bee957898f974c89646407c2addaa5762881fff6
50164 F20110218_AACMVY franco_c_Page_020.pro
6abbf481fc0085333c31c32aec78af41
99d5478e57da373679cb382913613ab756cf72bf
87623 F20110218_AACNCG franco_c_Page_040.jpg
634bb873d87f6e7c36a1a36baecb8d68
0dc81099e37bb70831d1de812817a2271a7ccd65
50573 F20110218_AACMXA franco_c_Page_051.pro
a2ebc20f63c47fafcc5d6d745f9ac7a5
51db1650713caff7b0dfabac1f5f50779d5b87a5
27885 F20110218_AACNBS franco_c_Page_031.QC.jpg
16d51928390d0bc55864e0a78118a22b
19c306090c3488a62ca9e4f1a57f7ce89fc43d57
51497 F20110218_AACMWN franco_c_Page_036.pro
562f81dbeef7236b9997ae34c504a7ff
4abb4dde99d08f3c173d8807cd48e33bb1599649
27666 F20110218_AACNCH franco_c_Page_040.QC.jpg
22e29881ea80d8d5f61409b4718fb155
740bac29da5faad2fbd72deb99145cb14befc67d
49164 F20110218_AACMXB franco_c_Page_052.pro
e3a77c1411ae266f6c1234edfdafb392
2778abe3a5ec6255566c7725e503245c0e66d5b1
86356 F20110218_AACNBT franco_c_Page_032.jpg
6730d687e5b2d9c1148a1c92bf26b7fb
3996a72c3b5648f0596e28dbe6c406add88b5b5f
50610 F20110218_AACMWO franco_c_Page_037.pro
3a3f6c4b6011a932ffef7fa8ee149875
7130a1fd6151e9a0b649e1d6f005d80afbc81c7f
48279 F20110218_AACMVZ franco_c_Page_021.pro
76d20f125a940e2e10256a205f1868be
c974cb0e746dd78099cebbb5a8e206b51c46d4d6
83878 F20110218_AACNCI franco_c_Page_041.jpg
4614b85dc1385f62761516859cf16112
b85c466de419d8a1ddecd5ff15e4ceaa7ef4f104
50549 F20110218_AACMXC franco_c_Page_053.pro
f87869bfdb1d5661afea3b2c089e25a8
d5f5131f3264074d224103437aa53b9e421b8235
26839 F20110218_AACNBU franco_c_Page_032.QC.jpg
77a178a2db1f05c7f5a7c0def5c8a389
fc605b6f2f42c0503bd7cc0df8c27d701840cdca
49971 F20110218_AACMWP franco_c_Page_038.pro
b7b1f0da2cd144ee98187579a15cf13c
479c9818395cd158f55e394309e37f952ddcf918
25912 F20110218_AACNCJ franco_c_Page_041.QC.jpg
339b2fa8cf3e0e9e35fc54e82087536e
d26b5553f9d8c1748c8dc0f4d26d17aa4af21876
46038 F20110218_AACMXD franco_c_Page_054.pro
b62bacaf45a8a362d302c3939b35da8c
ce1aa5cdfb44d1d317747a64a1d6635996347ffd
26300 F20110218_AACNBV franco_c_Page_033.QC.jpg
1fd91b2cf4e0520d61b3c3328157be71
19e380e82ebade06a941bbe387dc4013b82eafb2
46642 F20110218_AACMWQ franco_c_Page_039.pro
2e9c7d4940a28a28505bd53c0b41ecdf
7aa6e702c319076c61c8468022d64028dc922db3
84505 F20110218_AACNCK franco_c_Page_042.jpg
66e9ae2cba73b45b490573980a2001c6
77c75fe666419d398fe79de9faa88ec9dd6f396a
50937 F20110218_AACMXE franco_c_Page_057.pro
ece5077802a03f84a469f970111a2d2c
e4145f4ba70743a3b696c5e1b390cbc82a4b26df
23328 F20110218_AACNBW franco_c_Page_034.QC.jpg
6c723f1cda007e166ef2552a05a37dd6
0456858d5f052b121e9de2d95bf3c94b3be2516f
51705 F20110218_AACMWR franco_c_Page_040.pro
decaab3a5685720d21bde8bd406245f0
9a9d6ede3e55c2e966ad9054f4ce3041f6d2fe91
27361 F20110218_AACNDA franco_c_Page_051.QC.jpg
f72cfff6e6a90861303b6428116922f7
247d000348dbebea6d15113533d38f74d1376b55
26554 F20110218_AACNCL franco_c_Page_042.QC.jpg
7a987ea82acf39850a16f3e6950efb3e
78173fd8305c5a22909d57af64c55d5928c9ceb5
48024 F20110218_AACMXF franco_c_Page_059.pro
a0bb360588274bbb652b595b6eac48e7
4756f195a9302a364020b6e52678b736884304f2
82823 F20110218_AACNBX franco_c_Page_035.jpg
087d1006ced76ac4e79e3193f429ac59
62fee28ad77b17c31a65df99dfcc5c3ffafbb61c
51800 F20110218_AACMWS franco_c_Page_041.pro
0f5af8e1f63f11abe3cf88cc41ec5322
8cab04c1ffcfeab0a39408508100203bea3dad54
26361 F20110218_AACNDB franco_c_Page_052.QC.jpg
c70091b0b903116aafc5161be4679166
1216a72397605ca7af71c336afb5fcca5912e0b6
86874 F20110218_AACNCM franco_c_Page_043.jpg
0fd0eb8c4f2ad5800f11c99033a71f2a
ee1f8176ad40c48d18160f4ced4f0d209f49ff59
51745 F20110218_AACMXG franco_c_Page_060.pro
d63b96a2d03585f80166874d2f2d2695
09b4ce5e91548de9ab902eb2a36e57f43ee1c8f2
25961 F20110218_AACNBY franco_c_Page_035.QC.jpg
144242e5acb4d428c476e7388a686693
f0a3603f9f350c12c04042bdcb1f0ffdaa65bab2
50572 F20110218_AACMWT franco_c_Page_042.pro
40caac4200b01464a2f921bf6c77b0d8
c7df40008e5333f518eab462222e9da7d8fe2b49
83480 F20110218_AACNDC franco_c_Page_053.jpg
8f39abf1d7cc83916c87e94abfd98ae6
210497ff5540bcac9d2bee4a5a952432b13a4303
20859 F20110218_AACNCN franco_c_Page_044.QC.jpg
fd900cce0c9a447614d515cff4b842ad
ba138c808d44b851c763fd43545b12ddf87cab77
35244 F20110218_AACMXH franco_c_Page_061.pro
4421efb46153894591eaacf4493d89ed
848ea3a7c774af3593e1911d864ab36f24e0c263
84335 F20110218_AACNBZ franco_c_Page_036.jpg
1f47df1a5fe4833f4cda7bddad37d859
d3690136707f607a4943795616bb73e6595bd833
52215 F20110218_AACMWU franco_c_Page_043.pro
7caf9d7d7743701e23210764de5cd1ff
211ec1910263f09a11ad619f0d9ae51ec080d45e
25788 F20110218_AACNDD franco_c_Page_053.QC.jpg
8476b991fc7cefb2f18b18e557b62de6
ad4d2a1f93ce3c0945a69cc4599fb303a718a27e
43786 F20110218_AACNCO franco_c_Page_045.jpg
fc5741c5705304f1d0fca47f1b4ad922
3a6968cdc51ac21e4945c6106d4d759749ed8e5d
40027 F20110218_AACMXI franco_c_Page_062.pro
1565ede479f9ba4f9039fdc0bec9e8f8
d7421922b765971f5c8c1ffcdec920442a35436c
21624 F20110218_AACMWV franco_c_Page_044.pro
2978c8373219a2a77239bf609199f2c6
a97982b77e2db0228b4fe5c29d11645f7fe4a66f
79079 F20110218_AACNDE franco_c_Page_054.jpg
23a9a336136809ee723a6650da490791
f0230a1242d2d4729fbb97b03b87510f4a9020b6
13773 F20110218_AACNCP franco_c_Page_045.QC.jpg
10490ef070fae3583f9451b4a271a87f
6dd205042bd1986c5a8839910c8bf9218d10a120
21157 F20110218_AACMXJ franco_c_Page_063.pro
d1a8e207854d1121bfaa37dfe1c7670a
10e21fad5cce42d2a40fafb902119c264f6e453e
42984 F20110218_AACMWW franco_c_Page_046.pro
b5fad764126d83369872c3e4ed678673
bb72ce7cb3961fe4217c06c0097aeb9979b560e2
25062 F20110218_AACNDF franco_c_Page_054.QC.jpg
03d9e9daf86d3445e7f9ac37b679bccf
b6cbafe79cbf32ea270d79eb4fde7d506e1b3450
74852 F20110218_AACNCQ franco_c_Page_046.jpg
7ffd7b35a335be9ad6aff5e0c3347635
f668ac1b3b60de8cc0e1e2ce6886780885da30eb
46496 F20110218_AACMXK franco_c_Page_064.pro
556d441af060b9d356fd6de178e7a6e5
267884a3590f2b33d65e329b24b718d4ca13259e
51986 F20110218_AACMWX franco_c_Page_047.pro
a06a61f56a2196c3432759d2b0f387e0
df94c0304def9e04842cf63ab14660578be3d212
23376 F20110218_AACNCR franco_c_Page_046.QC.jpg
60126e79c2c6c8952cd3257c61a2154b
c577b878c7adf39fa002f6d9ed9712556eb9ff05
49293 F20110218_AACMXL franco_c_Page_067.pro
5856798fed31acb07116055665e024b9
51087997f4d342fe1ee93b829442502273f49f50
51667 F20110218_AACMWY franco_c_Page_048.pro
e22e5a1003f498cac60c9ca99a56b37a
ee88dde67bebcd36130c9f329f82a74413012b7c
89954 F20110218_AACNDG franco_c_Page_055.jpg
cdbe573c8a1d2047d98414b07c6107dc
da0a4ac9d4060a9633bd9f20bc69601f48282486
8343 F20110218_AACMYA franco_c_Page_083.pro
998686b6e567f522818338adf1edfd09
379ff4cd6aae138f83aa8436eefc7af9e9a31703
86895 F20110218_AACNCS franco_c_Page_047.jpg
d5201b6625be2cf4eec48d5be172fc7a
633d010cc6d396e5b0e049a33990a41326ccfe2c
50001 F20110218_AACMXM franco_c_Page_068.pro
938a331c93bfcff43c0e37c420f70783
5b31f1cf6bd216ee562f07fb9720401a2efe12f8
50913 F20110218_AACMWZ franco_c_Page_050.pro
1fad005719674125f992bb97f531a350
1699ee13b17801fd10cca908bee937bd2a63d1bc
28801 F20110218_AACNDH franco_c_Page_055.QC.jpg
7033f22c8dfa6d8e4bda4cc17a434af8
2b0959c25d3aaa13e581ef8df367771f8b45ed00
45268 F20110218_AACMYB franco_c_Page_084.pro
9fa201cefbcca7149e1486ab4a490bae
831a8d53edae55fce0de5312c7646f7237b990e4
86992 F20110218_AACNCT franco_c_Page_048.jpg
7fdb91daa5af201dcf5d295dfa979945
ecb1b138e562e0c05de285f60f3484c20d8e35c9
50540 F20110218_AACMXN franco_c_Page_069.pro
67a9d19b7a3a11f0232bda739ef90218
92b9072c967e73f2ba043dee7e73a556869d9079
80249 F20110218_AACNDI franco_c_Page_056.jpg
97161a350ff842cccbc79dd8585d4fe0
d7e1fe4ca5b427dbcb3d9519bedc9e393043b919
50479 F20110218_AACMYC franco_c_Page_085.pro
fc8063fdece9f4ac20e195040d07ba49
bebf3c5f0d6f8573c057cb44121ce30eeacb8763
27385 F20110218_AACNCU franco_c_Page_048.QC.jpg
36b4847b457d327075d305032d546a06
275798b5a0f3baeb00b0d02866db713aaa4588be
51517 F20110218_AACMXO franco_c_Page_070.pro
a929ede3b9b964711f6663cdfd8ba007
98b5d3df724d2cc40eed93860149d536c64f9fab
26315 F20110218_AACNDJ franco_c_Page_056.QC.jpg
423a8e1bd8d1cbd75c6a639e75f6b568
a2378791fd3e20721a055ba4952ec0ea9a3c72cd
50501 F20110218_AACMYD franco_c_Page_086.pro
7bd864dc689b79c35d396c882d0ba52e
64f75ce5a2d13bc64b40e7907db59e4dcd01520f
85823 F20110218_AACNCV franco_c_Page_049.jpg
a9262afab933718d965230be60338308
f414ae8deafb505435ddadecebed675379756bd0
53209 F20110218_AACMXP franco_c_Page_071.pro
6e906e77b09f4bdb7d82007118f8b82f
8115f4e0b1f1c3edc50468b5720ec436cb306a3e
83982 F20110218_AACNDK franco_c_Page_057.jpg
0caa8876002a041d9555fe188ce82952
586c4f96269233d59eb5a8f15abd7a52e01a696d
48949 F20110218_AACMYE franco_c_Page_088.pro
82aff86cd0cc9bafee4061356585799b
c266d22aaa85648e21340d44e67840e3d4123b45
26951 F20110218_AACNCW franco_c_Page_049.QC.jpg
9a843b62f6b3a054b5bcf61a36a3b65d
8c7d96ae1e992d62b73647089d7baa2f272b75b2
43152 F20110218_AACMXQ franco_c_Page_072.pro
b33ce76c71df7c67be5a19dd6f35994c
309e0daf6f617c9e1edf124f1d3ddbc48b919e51
25954 F20110218_AACNDL franco_c_Page_057.QC.jpg
09b576ba5be9619479e9fbd065900769
35733b741ebcf660c87743d7d7684ff5fd18ae1e
47484 F20110218_AACMYF franco_c_Page_089.pro
998bc4fab4fb81959c6a973daf014436
998e0c28bb1d523d245da82424a068b3b759553f
85328 F20110218_AACNCX franco_c_Page_050.jpg
e554f4a236d15dbd17598c334f93b96a
08c9b56f1f4f322732ce3cb21f45c95d96f4f7bb
51507 F20110218_AACMXR franco_c_Page_073.pro
ce3655958d1faf4cfb728f7c074cde92
3cda9b97ea9ff661afb3021937be0d39326ec872
83616 F20110218_AACNEA franco_c_Page_066.jpg
9302bb6d9ffedcbedf31c6e1a87aa464
cbeff3b91ace6ac1062ca0bfcb054a0753eb733b
86559 F20110218_AACNDM franco_c_Page_058.jpg
f240d3eb3ba6cdd085ef4dc952a9782a
c0b4f6549aab26f917da4a3d0f11b7932f7fedcd
51725 F20110218_AACMYG franco_c_Page_090.pro
b9e4c2f9af59dda7236f733aa246a6aa
e0c16fce8bfb64e35064ae33b62bdcae1864aa2b
26292 F20110218_AACNCY franco_c_Page_050.QC.jpg
47ff054ddd0594c2a496b768b8f9ff4f
b12a3da058ccc326888cc0eefa2ae943c97e5658
50550 F20110218_AACMXS franco_c_Page_074.pro
610484773f6f01cfd5403df68dac4899
2748fa5a2508859b32530558f09c94b071d6c4e4
26229 F20110218_AACNEB franco_c_Page_066.QC.jpg
b9d0af990bde893b74e09c26be8595a0
e8f20299a32ae29c57343bc70fcb70bea80897e5
26797 F20110218_AACNDN franco_c_Page_058.QC.jpg
4c9b08acf1f22ec770886ec23a1981ee
c9900a72afe8e7962e3402481abd9e8cdae544d2
49149 F20110218_AACMYH franco_c_Page_091.pro
f1c051a098d02360434a68ca218146d3
839166f3e3d13a0433f608b2a37bbb29dcaef6d4
86655 F20110218_AACNCZ franco_c_Page_051.jpg
8f6be6af2601103ec043974bda1ea687
aa29480ab84e222be4cc31d0a58649d93e303b7b
52064 F20110218_AACMXT franco_c_Page_075.pro
ac07725fd0e37ca1ae93790f032c15ac
e1912be799f931d0fc95eda3d9b4cf517efec73a
85044 F20110218_AACNEC franco_c_Page_067.jpg
d24f7db400275ab581db40056c1686a5
0852bca7ac9675756d3e01eb6c03ed4af4384cc9
79308 F20110218_AACNDO franco_c_Page_059.jpg
952ba8a62d22359f539d54f27747e7c2
9640f9e22d5ae7ad6444c4bb21b4dcdd1c2a2084
50433 F20110218_AACMYI franco_c_Page_092.pro
ae773426bcba394ee3ae8cc8e55ddacc
529575fdeb5c217766c8c8794c7f4e82febde65f
53259 F20110218_AACMXU franco_c_Page_076.pro
54ef620d73d2a5067e132f549f7c01a5
b9a3f9ad23e7e7f29b7d8c6e056982a0f2c5bb35
26718 F20110218_AACNED franco_c_Page_067.QC.jpg
85d96515036e60d3f92ebe36253ddac2
5aa9905193de5a064dda730ff6ec0d8bf9411611
24816 F20110218_AACNDP franco_c_Page_059.QC.jpg
ced317788ce583b838d4c8ed0c8acc5a
0829d1be30a0bbc5f9475475f4247da2e7ec3729
33918 F20110218_AACMYJ franco_c_Page_093.pro
51e5d3ef202faa96109af15421ada404
bdbe12779786254ef8e819c86be36fdf26e4ee1d
52578 F20110218_AACMXV franco_c_Page_078.pro
97aa31499317e2e21cc6270b679f4e6f
bfb6cf1fee503e86bd67d863335f3bbfd05aecb5
85628 F20110218_AACNEE franco_c_Page_068.jpg
738d26b95a3e2d0fa3275d224e726c9b
859aa2070a47d50892e8a4ac7e64497c78429192
84282 F20110218_AACNDQ franco_c_Page_060.jpg
49f581c21d45d254d5fe3a3afb629311
dbebeb59dc32f2c7341943fdc4af975364d53a8d
52370 F20110218_AACMYK franco_c_Page_095.pro
dbeb8e948350674bfbe10bebbe791a72
0995c02bb5c50c71bcad6fd853fc46ee7f015cca
5380 F20110218_AACMXW franco_c_Page_079.pro
9992c5fc07470617b82167f63e14a0db
15eee2e4c49798d1630649891fc6f4c6dba2c597
26606 F20110218_AACNEF franco_c_Page_068.QC.jpg
0023843baa26f55e6c0e6c4e00446984
0f42022c0a4d578fba5a254a97710ced8702bdd8
26105 F20110218_AACNDR franco_c_Page_060.QC.jpg
78bd085d455c20ee237306843d84715e
8ae5989b63e3cc0233aea0a6faa279b5323ab4c0
48478 F20110218_AACMYL franco_c_Page_096.pro
fe82c623b6c09922ebf89d2e90f0591c
8925e889c3bdf4c0c42bcad28f7285d08fd2c297
50654 F20110218_AACMXX franco_c_Page_080.pro
81573b77f1265fc9f7e8e49c8467a377
ee2b83bd09886fff0ef7e692a733bbac010dec68
85758 F20110218_AACNEG franco_c_Page_069.jpg
6ce126ac15f130d6a7e65d324b3f8e9a
1af597653029003ab0f7b7bec1e161c6ad50af8f
63823 F20110218_AACMZA franco_c_Page_116.pro
2629883e501d31c26fced2dc4f35056d
04509a892857188e26cbfe82e0a4ac64cc2ba223
61751 F20110218_AACNDS franco_c_Page_061.jpg
f77837b68f44ca0e0d038ce3ed5dc4b6
9ce2566c38433de8c5d301c50de5a8501b84d3a4
46657 F20110218_AACMYM franco_c_Page_097.pro
5cf854d8a816f228c3b1a2156790c588
d6572677ba3b888f57ac2a8d9196ec61807dad33
29737 F20110218_AACMXY franco_c_Page_081.pro
3c4920d376d35db825313ba623bb7cff
3e3e4236f169739cd2eb3e70835ad4c4b0a578bf
73241 F20110218_AACNDT franco_c_Page_062.jpg
f97868110eae1d43dfe9fb7ae665e39e
d27a73ad7ba68685eef5c40ef05b9728f0cf0773
48495 F20110218_AACMYN franco_c_Page_098.pro
37196c8e355a614ccd128778faf83b97
053fdc394ff9359b71b16e51059e6807a4f36885
32871 F20110218_AACMXZ franco_c_Page_082.pro
3e611745a9f8d6efa34c99eb4aceb515
70f4bddfde72c376ed23ff0c8a33f5f6704656b0
27894 F20110218_AACNEH franco_c_Page_069.QC.jpg
a00ebce7b3c61a95ecc3f7c83c7b5c9f
adf1e2ae34f1b40cb9d341e621bc54e4fce5a911
61305 F20110218_AACMZB franco_c_Page_119.pro
a39b19b162332830bc50e07da2556bc5
41d23914e8f0f37452a2f1256f033f920406fd31
39989 F20110218_AACNDU franco_c_Page_063.jpg
b79e277a333ba7bc9ca44be0df00d7b9
c1a4a1ea3e979e4c94a92bd62139eeb633624cc1
49560 F20110218_AACMYO franco_c_Page_099.pro
26c89ee80b7adaec269fdf6facca7fbd
a4a5212d9e94b82c7ab8ea4988e1afd44469da0f
F20110218_AACNEI franco_c_Page_070.QC.jpg
f0211afc9033366c1fc5bbb3af78689d
fe026da07bc68d85fbc9d835eb79d9297e1ca839
68980 F20110218_AACMZC franco_c_Page_120.pro
4614db3252ba4a71dd91270f1a182801
e30727603daa5284912c0c6a94aae2947fd81eca
11392 F20110218_AACNDV franco_c_Page_063.QC.jpg
279d58949b00b3bd0be830cea0646c1a
d88e139379158277257d1c526caa25a0e9180281
49435 F20110218_AACMYP franco_c_Page_101.pro
727d36b562938ec839b52882040f5b56
e0deef19657dd023cde7aa2bd162dce859d5f339
89937 F20110218_AACNEJ franco_c_Page_071.jpg
2272f6cb1de4ca2d5ca8eb8d8490a781
2630f10eb72d0ef77eae1159be5b3fb5d32cfde4
63296 F20110218_AACMZD franco_c_Page_121.pro
bb8a697611e30d84d9345352f75dbc0e
a306f1b7c7fa5e8e06bc9fd443a85805f182d74d
81814 F20110218_AACNDW franco_c_Page_064.jpg
07072a5e6398fc2030be688e45673ef1
c748544a517b514a09dd3b38b955e06bd964b402
7512 F20110218_AACMYQ franco_c_Page_102.pro
95932415e7bd30910fb59cc78d165998
166c08a62341d2b0cd88465474fd6e98552d3252
73402 F20110218_AACNEK franco_c_Page_072.jpg
48b3fbf95c030c0a19a4543637fe1283
b6e7f68a828c270041a8530ff40d5cfdff4918b7
67261 F20110218_AACMZE franco_c_Page_123.pro
19548d8bbb9e1fd7088674c291b4d112
e031decbcf2ba69dedbf8f219069f740fa0ac72d
24778 F20110218_AACNDX franco_c_Page_064.QC.jpg
e09172dc7f307fd642a33a2424f75cf8
eb5b3e0c92431a25c4dcbfb0d84cf754000239e6
60403 F20110218_AACMYR franco_c_Page_103.pro
9d952e290f43c4ee10b54fa311e256f3
645d64f02d95bfd3a919f0c766b85fc53a238d3b
53320 F20110218_AACNFA franco_c_Page_081.jpg
aef188fb3ab00725667833e4eb1c4557
2b13670086b8b9c4919048c36ad6d9b3005ea5f3
23649 F20110218_AACNEL franco_c_Page_072.QC.jpg
32315cc92dc5c0e0dedbad45b1becc69
6aae4b24355b9b1cdada1f74a6dc89207675b1f2
61784 F20110218_AACMZF franco_c_Page_124.pro
c29d8a8bc8f0cc9397eda481b58e4340
c83ed2565629ce6e0f565930e3b52de2be1067a3
83621 F20110218_AACNDY franco_c_Page_065.jpg
4c34f58ffae566b010c3d66c6a20ac7c
d681c69d3b670c27545a5e44fd2707788b74ce2c
67362 F20110218_AACMYS franco_c_Page_104.pro
1442b35139c7372651125e23acd77f20
4dc35c6f6ac8061994daf53eaa74ca468cafbf7c
15201 F20110218_AACNFB franco_c_Page_081.QC.jpg
79046cbb29c007fd0a298bd969d81e73
d8a44417568bf55769acfeea617a0d5d39dff694
85387 F20110218_AACNEM franco_c_Page_073.jpg
fd19b477ab3e6c8dfafb1e0b41dd9983
ad3eae507f4a3f048f90fd0f9bd60e992cb4bd36
63081 F20110218_AACMZG franco_c_Page_126.pro
3d47f3e016184077ea7f73a9555aac07
7e46f1de116dee8dffa941eb39cdbfb242ada293
25726 F20110218_AACNDZ franco_c_Page_065.QC.jpg
b3604cc7d6db4ef67f14d97b854e11fb
31e5c0fbdc1a8459c324159451c6a513febafe01
63162 F20110218_AACMYT franco_c_Page_106.pro
fe3983f3f06e63a5b996d740da0280fd
b2d5a2ee120d575224df90ff69f766bad903701f
56530 F20110218_AACNFC franco_c_Page_082.jpg
015f0eed8b92ddbd67df351e50545cdb
2812bd445d60dc2d0077344f79790b97807f43e5
26414 F20110218_AACNEN franco_c_Page_073.QC.jpg
816739436d320224022b4fbc64217396
3e06bdd1e45d3d4bcbb3ae55f9ffba3864113d78
61606 F20110218_AACMZH franco_c_Page_127.pro
4bf1aa97bae48f79b3b687e5e342625f
bf89150e9e9b331cef6847b90848cdf027ac4901
57841 F20110218_AACMYU franco_c_Page_107.pro
b38c1097065bde29927a26f4f42d3a86
2e0c924782fa60982d07e6d39faa04279269af85
15611 F20110218_AACNFD franco_c_Page_082.QC.jpg
8ea08372e9c051efac6634485f58bace
ec7795fa635d180fcef23995c2d5cea89b298186
83758 F20110218_AACNEO franco_c_Page_074.jpg
89e2dd787261e25fedce73d07e382d5f
4bd38cd4629090e1613a03dc92048b25f8f4c590
61713 F20110218_AACMZI franco_c_Page_129.pro
ab94698f9516d890bc1b2f823e0eec5f
c7d30da9e9bac12c83ec8c68c19a703d45c743a5
61008 F20110218_AACMYV franco_c_Page_108.pro
15551e8eaf4253f015b774686248981d
fb9f7a5fa406feada9e7bf29c6fbbd0ada2a01bc
37366 F20110218_AACNFE franco_c_Page_083.jpg
5c189b3f93c2b08cd437e814483a071e
37a097dbce3a3800ccbd10a917ba44c35efcab8e
26679 F20110218_AACNEP franco_c_Page_074.QC.jpg
f0cf3d64fec73ee77a34f557db2f2c67
bb3429e64edb918ca9f5e0205ecedc5de9a8d42c
62313 F20110218_AACMZJ franco_c_Page_131.pro
fc8e1e46f044ad601afed18915b65aaf
37556364f07d260fb9fd260baea86d1d3daae45c
62306 F20110218_AACMYW franco_c_Page_110.pro
a311069e82b5a873a0440b9a8de0ff79
3aff3eb210a8616f0fc5576adae7f015f947ea6a
11493 F20110218_AACNFF franco_c_Page_083.QC.jpg
bf1c5812e6724310cd55af0fefdf4e78
c045380aec5e0748a5cd25a7e1e81bac567bf497
84699 F20110218_AACNEQ franco_c_Page_075.jpg
7ed3077e648d7adc980405b2a907d610
3d6822ecd7be5e1b1091251e935118a764f28982
63408 F20110218_AACMZK franco_c_Page_132.pro
73ee0ea4ba516f64b3c85938706c4b59
9b4a44d153b600e1f744faf0ae73c5ec05ece1a1
60546 F20110218_AACMYX franco_c_Page_111.pro
4709849a6cd47d8943ca6ed7212c38d4
6f5de57fc6bf1dddc78d7e2ca31ff74261e6abfa
83032 F20110218_AACNFG franco_c_Page_085.jpg
59f592e30eab54f1726fb60b797e44fa
40af1d8edd31506c1dfe143b362c84f0259d866c
86986 F20110218_AACNER franco_c_Page_076.jpg
c07c901ed8bfed6b5b216d6723a44228
e89ee1410b156dab39047ad9f614953da31076ee
47913 F20110218_AACMZL franco_c_Page_134.pro
7c1447c2e1cc45439a1dc688ffe1d226
e49ff725541e6ad22c47d2003c4ba39e1ad0e039
58450 F20110218_AACMYY franco_c_Page_112.pro
66c558d502d921923208507cf340ad1f
4db8f64fc4e0eef2064b0f2fd1406d79b97ef6e6
26020 F20110218_AACNFH franco_c_Page_085.QC.jpg
084690eb4c626915cf1e6aa53305fa18
4b353edcf617e1b4e955567b9fe20b1fee1a53bf
27120 F20110218_AACNES franco_c_Page_076.QC.jpg
81f4778abe2d5a602273c6b88762be37
0c1cd0aeac1b7b5b197b493a659e805633beb7a8
30186 F20110218_AACMZM franco_c_Page_135.pro
8848e27833f8e7fac71175f55ab09315
efa2ffa58e4df173a9528ec2712772e0e563a238
60806 F20110218_AACMYZ franco_c_Page_115.pro
3ac009f4c8ada96147aa247a24698cbf
319ee12bea4b46fcc9acd2f36e13aa77afe14e12
83150 F20110218_AACNET franco_c_Page_077.jpg
e53ae8528f49c99245580041250cf3c6
8f55e26ff1e7392806a3463c99af0920fe4832f7
23960 F20110218_AACMZN franco_c_Page_001.jpg
1a7e67334d49f1b25fd619bdfd2d4df4
bded7a411a9f017cd4d357a7268ea49172afc7f0
85403 F20110218_AACNFI franco_c_Page_086.jpg
70702f6376ede4e75586bb5646d80af9
92d351c677658055d66a9ea8df3a6cd41a34cb10
25798 F20110218_AACNEU franco_c_Page_077.QC.jpg
d1e174203a0ee9824d20ff3d744c1379
ff7583d72bd6122fc82024650ef59baa890b9e69
7308 F20110218_AACMZO franco_c_Page_001.QC.jpg
68e88f1e273f89db5f3341ff1e3e1326
e6a1e1ee4407ce1f5fb354fc041732f71a470fdf
26384 F20110218_AACNFJ franco_c_Page_086.QC.jpg
a3415b8db8fca8969d554c74ca99354b
8c1d96275d29fb5c0503ce8d1ee5443eca864525
27363 F20110218_AACNEV franco_c_Page_078.QC.jpg
55218fe111c89b8d554ae0bc65cd7981
3c9f98d956497e7ad3a4b11fd4ab4594a61fbf3e
4312 F20110218_AACMZP franco_c_Page_002.jpg
37e6f81a1b9a1d2e638dece56f0958c0
40dd234aca4c8e0a045f7cb7dda5d81b899ff696
86633 F20110218_AACNFK franco_c_Page_087.jpg
49bfbc908145fc0715e8561a9e089293
14e17a380f398edfcd4c3a2040688a878daee0e0
11788 F20110218_AACNEW franco_c_Page_079.jpg
8ce4f31f859a7b0d4cb7fd04c6bb50a3
1afa00b767a7498dd227ce40f31c84f5c6b141ba
1399 F20110218_AACMZQ franco_c_Page_002.QC.jpg
b6dc575626577b6571cae86321cb5f12
6bfbb3bda50cee6949456728c62765f6d708542e
27156 F20110218_AACNFL franco_c_Page_087.QC.jpg
b18c82051fa92b840178016073741d17
0d644970e6507bf09812ace33252206b7d680f81
4214 F20110218_AACNEX franco_c_Page_079.QC.jpg
65db14ac62dd766d0441d534ea1027b9
8af40cae6e98cc03d737f3cd62e8eda323c88370
27124 F20110218_AACMZR franco_c_Page_003.jpg
e5c5e53ea735bc79c236c7096d38ce3f
c71ee3f4b384561d3698de0f1903b9749a087532
77801 F20110218_AACNGA franco_c_Page_097.jpg
70f13637134709106bc5a5a16759ceed
ac6ce3df69ee1051e34975e40ee1ac7928d3adcd
82350 F20110218_AACNFM franco_c_Page_088.jpg
07f001d5da27bdc601c3a413ef548800
4e2a0436093cf65b0ad56123b27ac6e98553a263
53327 F20110218_AACNEY franco_c_Page_080.jpg
f8cd8011eb0812e203a67a558ed53921
a0508cb303176509ddc01d0a3713ddb5010e2638
7522 F20110218_AACMZS franco_c_Page_003.QC.jpg
7732bb857d9f6fe55b03b75f40938a5e
1605014656a2d85deb0087dbaaa66b9c8225b1d9
24020 F20110218_AACNGB franco_c_Page_097.QC.jpg
97d46e70a0ad8c08c061acb02d533fcb
8687b69fd2ff6fb719e63855b10c49bd3b28fd32
79482 F20110218_AACNFN franco_c_Page_089.jpg
83b716b1804a334a151cc48b2cdc3a88
0535e716bbcbd3e938d99a4cd33d40c44c933aa5
14011 F20110218_AACNEZ franco_c_Page_080.QC.jpg
e3144a56db05e6bb0c19ca06e3778eba
ce6a15c1d7bc817f70b69acd4ff3ce19d3194a26
69704 F20110218_AACMZT franco_c_Page_004.jpg
775eb0580e129dc64ffff0a59b4f3fa2
35dded827f69fc8e03c7f78870357c38d501ad3f
81471 F20110218_AACNGC franco_c_Page_098.jpg
4e7df04b024f50fb52d9f16490dc96c6
da51ab8076d6df85409d80d48f930392ec8eacaa
25064 F20110218_AACNFO franco_c_Page_089.QC.jpg
4819e917cb755fb66335bb9296c165bb
511b9a451a73b8eb15270065b72c2578adfa39ee
21641 F20110218_AACMZU franco_c_Page_004.QC.jpg
6568342fc6f4d783ac09cab5c6ddd6cf
88af33db09261d5638ccc55e33b2aec15e02ecd0
80370 F20110218_AACNGD franco_c_Page_099.jpg
ee36d23a7572fa1d10f849544cf8117b
3697387780849e78281fd5cc384adc0cbbf74cae
85065 F20110218_AACNFP franco_c_Page_090.jpg
0464f9c507c185d0a72ebcf82cd84551
763cc0cc1865b8a4fd6907b3580f90ac4fb93d29
77608 F20110218_AACMZV franco_c_Page_005.jpg
1dcb50c38c981f1887be6a62e7ab7437
717249aa12294ddbd4dad3062f4cdf2686679f3c
24815 F20110218_AACNGE franco_c_Page_099.QC.jpg
404154dc9c370e96c28b211143d3c3c6
ba88a7b69621d44bc8eeb0b27a26d71fc8b5b3d5
26446 F20110218_AACNFQ franco_c_Page_090.QC.jpg
0d8e0ae92b3b7773ca076fb73d774747
3bec52f1aa95374de3ed718c32223d6ea7f96bad
24266 F20110218_AACMZW franco_c_Page_005.QC.jpg
c5451c056bd51dd7730f1bf3e7291295
62dac5c7cf15b91a90c158d76b6f9ec7b5f67a8d
80376 F20110218_AACNGF franco_c_Page_100.jpg
cb9f3e6663e899d12514f44928b0c3d8
cc058b0ecdbc43094f28b81e44329654161ec37d
25177 F20110218_AACNFR franco_c_Page_091.QC.jpg
0a697c6b4a4977d31fe2bbbd76d644ca
1286b0381d360beb159e312df801de0d4a09da5b
70449 F20110218_AACMZX franco_c_Page_006.jpg
27a6acf22e6953ae528ad5f2fc95a614
416cb80ff10a50eabda68ae2de859f055382043c
25250 F20110218_AACNGG franco_c_Page_101.QC.jpg
20c72795c4753dd969794a4d1df6e95e
3df579e818f4b37b596d3952538ca677ce86053c
25996 F20110218_AACNFS franco_c_Page_092.QC.jpg
c4be634fc27b22228bfb838bc21063fa
aecdbf92ed8faf58c0f3dede69d741012134139d
16088 F20110218_AACMZY franco_c_Page_006.QC.jpg
0d022e9d02309dfa0395072ad1541fdd
c80ed61dee56a4a50a13f2c715683937903bbca9
14875 F20110218_AACNGH franco_c_Page_102.jpg
08b2834c49cfbf41fb4fdef07635fb77
7c2a27ec29199a619f4d8a2855f3d99753fccaba
59242 F20110218_AACNFT franco_c_Page_093.jpg
7384dfaad762285e141cb2459aa586ff
b127a89fdc50800676b593153c94c93a3602f1ca
110519 F20110218_AACMZZ franco_c_Page_007.jpg
a427880e316d7f0944a720d0da54911e
4e8f1d7fc25531f07b6d9d44837faa5f84b0e175
4985 F20110218_AACNGI franco_c_Page_102.QC.jpg
b008b42c691fd62fa122b665a0814472
7026d57afde167c7214014cb66253df4ca0fb90a
16831 F20110218_AACNFU franco_c_Page_093.QC.jpg
f1586129d31af128969cd2276bb5b2db
e7ddd4df8b7493f6e25e0e50f6c3ddb7552debb9
79151 F20110218_AACNFV franco_c_Page_094.jpg
26b1e993913d45d9ae0830202691130d
b53cb01929467e0a4317041ecca0eb6ec8df999b
98336 F20110218_AACNGJ franco_c_Page_103.jpg
c883d3416c3b84cb4a101215dabc31e9
7f025e496b7738a039176487b377a2b346feaf72
85729 F20110218_AACNFW franco_c_Page_095.jpg
8768abfd7ed7ab1c9ef7b24c11a11e5b
799941c8a792704983724e0eec96dce5252b7061
110162 F20110218_AACNGK franco_c_Page_104.jpg
bd590f58cb2f959f9b9d7977c96fe524
a72aa5050fe2083125bf479c53fab6ae923a9e05
26803 F20110218_AACNFX franco_c_Page_095.QC.jpg
87c729b43479de9f4d9c464bfd518c60
bd165675185bda160761bf0df8a8ec07ce008181
102304 F20110218_AACNHA franco_c_Page_114.jpg
856eb97758c50122e946895770544acc
b0dd09305e84fcb6e214209653e91aec7304accb
29655 F20110218_AACNGL franco_c_Page_104.QC.jpg
72157e1c1849bd61638cc1a4ffdceb2d
afbcd5629b492b5858b8fd1308911a2d56eaf371
80992 F20110218_AACNFY franco_c_Page_096.jpg
4107d8548742642d7e617ad599616b5a
5bd543b4c7f62bd7fe3361800660aca3a18c3705
26978 F20110218_AACNHB franco_c_Page_114.QC.jpg
57863f948fc012f21ab543124e5df0e6
5a9eaee5d1a3c09ffd85f08de7a80d7f65ca3454
110783 F20110218_AACNGM franco_c_Page_105.jpg
08301321a5f9b87e5d5504eaa4b64fd9
dda75c2331ee77a55e9185a04a8d806ad8f669f9
25340 F20110218_AACNFZ franco_c_Page_096.QC.jpg
911a2356a1e6a41474d46c48cf2c4397
d45388a34fb6855c7e0ccfe8d2c2481b18640a9d
107335 F20110218_AACNHC franco_c_Page_115.jpg
cf8adbcac0924cb8de8ab7c3c43d4f51
ee30668747d1b84b48a6236b9c129154d405c1bb
98007 F20110218_AACNGN franco_c_Page_106.jpg
8388797da6d430a7125964f2020a36ab
3ca7930a2ad04ec918706a88a8d00a8e9f68b1c7
28416 F20110218_AACNHD franco_c_Page_115.QC.jpg
48c07df6e35639559676bcc0aae145ce
ed0f86ced6389730d49a269de14c98b33f053a40
27828 F20110218_AACNGO franco_c_Page_106.QC.jpg
61e5c8dd858d305af493ea029d274cc4
645852a797d03fba9e0c6ea2a28da7758d480467
110574 F20110218_AACNHE franco_c_Page_117.jpg
30b4bee1309d7e5eb8dcea98c2cc9e3c
fa6fd5604e7c33886b684997b182a3bd12f82f7a
95152 F20110218_AACNGP franco_c_Page_107.jpg
fb1c3514cb278f10a74382ac4bf3791b
bccd80feb82f88ef79d738307c089b049eabf92c
29307 F20110218_AACNHF franco_c_Page_117.QC.jpg
bc4cc0340ce4323738d3e69aff0bb1df
9933e182d08cc694e7c1c61302126a75c01b83f4
26546 F20110218_AACNGQ franco_c_Page_107.QC.jpg
3f3bde037ccf1727f6f4412db4acd423
bbc4c26563c4f0d0249917e2012b25fda96f040b
96781 F20110218_AACNHG franco_c_Page_118.jpg
728e5699f3c6959e6cdcd95214833f17
764d3fb200a07ecc45369a45c5180858d1a98630
103963 F20110218_AACNGR franco_c_Page_108.jpg
09a82565aa2cc2b7fb60cc4cec814dda
efcbe1874ae5be6312d8edba08d6dc5948ecb73b
26778 F20110218_AACNHH franco_c_Page_118.QC.jpg
5f1b2353e7ccb9976d2ffcc5280699d4
70c9640fc568de2a9ca9601a54284a14f36bc50f
28126 F20110218_AACNGS franco_c_Page_108.QC.jpg
e9b699e00b437954a060e66bfc19b448
443e3544c293a6b35f74eb2745376c3091b855ec
106845 F20110218_AACNHI franco_c_Page_119.jpg
8f7546ed602b888705d22ac7861c6080
8618083d6459803fd8807e2e8b1fb8b25c6b7e21
98395 F20110218_AACNGT franco_c_Page_109.jpg
ce782f923a5de9f7081538968bdd82a7
17af60ac65e460c93e0ce824c3f5460113757c33
28707 F20110218_AACNHJ franco_c_Page_119.QC.jpg
a2e4f34d9ff5ab35e4831c1b6de99e5b
4444717474f9f9b4cf61dd08845918c308cc034c
27623 F20110218_AACNGU franco_c_Page_109.QC.jpg
d0304159d5e311e2330993313027f6c3
33650cc8073f01eab451dfaa4f9f016c8e5c1b0c
105150 F20110218_AACNGV franco_c_Page_110.jpg
af31abbcb3d9c42cbaf223c3cb1ca891
f239f52d22496b109712038d94c19c677f5949c3
110029 F20110218_AACNHK franco_c_Page_120.jpg
ca2875a0a8f1f71998808ee7e124338f
9ba69724df6d2749e0ea56215e6e80a2294aa4f1
105506 F20110218_AACNGW franco_c_Page_111.jpg
6a38c7c50a28e8afad53a0044321eb6b
fed77c074a8949f24f44ee5955b694fa3f241541
27816 F20110218_AACNIA franco_c_Page_131.QC.jpg
1c8c5016beea748bede397053e80a9ff
debf486d1f9e74ff49e0160aebdd39297f530f45
102692 F20110218_AACNHL franco_c_Page_122.jpg
f3bf2be43b88025d94bfd0eeb7f7976f
0661730c64704e1d0c980e46a457acfdb7c42ccb
101772 F20110218_AACNGX franco_c_Page_112.jpg
e2db379f2ad9d303e4faaa70618bb218
5c6366f5d2be1710e0060ebbf62670280d431db6
109273 F20110218_AACNIB franco_c_Page_132.jpg
998ce2c304a6e820dbc325294ecd0809
7ada6ffce9d38ddf7341b732473c4651abb1821f
28330 F20110218_AACNHM franco_c_Page_122.QC.jpg
a084bba14d2d0816c696b058edad1b79
693a2fe4899d6066392cf7d19c7f7827b16b558f
27588 F20110218_AACNGY franco_c_Page_112.QC.jpg
a6709597a1dcd534c070675a81d21ebc
6cd8a0f7f91a4b4c4baff96e8f2278715e5c80f8
29056 F20110218_AACNIC franco_c_Page_132.QC.jpg
177ae361c47d3b3327585a2c5faab672
130fc72981e6e91201947674019cce87df4f92ed
113044 F20110218_AACNHN franco_c_Page_123.jpg
6e39874615cedf748dd7b2eff92148d7
cb5d412423a3c7fc564044c228503607c3a8889e
103001 F20110218_AACNGZ franco_c_Page_113.jpg
b6d2574cb517e3f40eacf92c70320100
bb8ae633852c2143472bdb97ed29b1150ade945d
112388 F20110218_AACNID franco_c_Page_133.jpg
ccf596f61f43ba4ae00935a895b38405
cd3b0bf5dcf10a629fbb0169325a75aedcd0b2ff
29338 F20110218_AACNHO franco_c_Page_123.QC.jpg
f90d171d523bf6fecda40c33f2cd74fd
8d8d0a16bbba8bf16ad0f556c3c2cfc72711e1e6
30256 F20110218_AACNIE franco_c_Page_133.QC.jpg
5b093a167211a88faefd2216452d5f78
22a01987b6e72216692082dbf1f0e31dbe8d09ce
110037 F20110218_AACNHP franco_c_Page_124.jpg
bb48335889b1da3143a6385b2935aded
37a811dd59a6a40297caa12e015ad03839ecf9cf
84520 F20110218_AACNIF franco_c_Page_134.jpg
42a44a0d1f9b053cbafd6f2c126a6e5a
eff3e85c68fbcab823e083b52682dba24f501ef6
28739 F20110218_AACNHQ franco_c_Page_124.QC.jpg
9b5e6d746e6c0b0abe483bcd2354c7f3
76e31793fb48d487c0edca3d80a44cd624d344b6
23247 F20110218_AACNIG franco_c_Page_134.QC.jpg
9902a01c035575e5426e230353186dfa
6b9f4bf064991f81e6301f109766cc5203ad1a77
108111 F20110218_AACNHR franco_c_Page_126.jpg
065045860325be9cf7621c642336dd35
42bc49dbd2cc231855122d02e684901fd51575b7
54288 F20110218_AACNIH franco_c_Page_135.jpg
775d1f5fc35e33a4df7793f910c7aee4
bd3c0da6cdc6c5dfde8427ddf879ca7df56254ca
28633 F20110218_AACNHS franco_c_Page_126.QC.jpg
b61c9c190a2448af6a6e59a31ae6c458
524e778fab564602d0304feb339227b016928e5b
16950 F20110218_AACNII franco_c_Page_135.QC.jpg
b8829421b7641b77b24de957f88f2dc4
15fb88c6f324d2d232e553c5cd67dcdef99e13b3
98713 F20110218_AACNHT franco_c_Page_127.jpg
3c57358993f5c4f9e1fa68df4570f7bb
bd0f3619884474b4c545966f8a82f09ce60e1c68
271967 F20110218_AACNIJ franco_c_Page_001.jp2
8800afbd436fafa9c54118196be887ca
1e9f160c7d10d3cbaeca2fdf311025b7f9ce2cf0
27248 F20110218_AACNHU franco_c_Page_127.QC.jpg
78327827eaaaaf9f02ba49c2f739becb
7948fbd37730faa2fb774460a766921fbf925128
27866 F20110218_AACNIK franco_c_Page_002.jp2
3686263f97209507e63f347e31e3a1fb
2a1285fe21889499175a019f1fed4957564e8992
106094 F20110218_AACNHV franco_c_Page_128.jpg
114f098e5a9c8d8c39d0ee848b51a27f
69a5e81fc3243ec054b8cf79d2e9b58ec86ea6af
28864 F20110218_AACNHW franco_c_Page_128.QC.jpg
f25e5da86a6cfea52215edce38f9e19f
71040580783b1e1c9beba15a460389acd3dfbb9d
928647 F20110218_AACNIL franco_c_Page_004.jp2
3bdccec71eda94eafa8a73e8d9a9b2fc
678928216baf4f9090f5b29dcb9a351c7676e8ff
97742 F20110218_AACNHX franco_c_Page_129.jpg
e0abc6cc4a10a001eadc55a76c900007
a2f5c1097db9207a5d2f8f28a6c2d727c78ae4da
1029982 F20110218_AACNJA franco_c_Page_022.jp2
91bc1f69d9db04dd0ec84aa2037b678c
61d4f6204a958c4df67ecfb7b33be33e0a5d82ea
1023122 F20110218_AACNIM franco_c_Page_005.jp2
86766348114d6a2698196cef9165321b
c49bf86b3b3b5c385797770cd1c75ef3cf1c6600
27602 F20110218_AACNHY franco_c_Page_129.QC.jpg
69126dbfbe3e6d808ddbaad9caefa862
17194842312d450e35c0b65f48053631bb34732b
1051912 F20110218_AACNJB franco_c_Page_023.jp2
18894d21a0344b4cb68a29b8ea1eff28
d4fffdb1175cac65021293b8925c75b59b25e3ac
907943 F20110218_AACNIN franco_c_Page_006.jp2
35741c2424986551729a04269dae50e7
1ea01b4b1b496ccf555e7f75812b64e62b59a5f6
28396 F20110218_AACNHZ franco_c_Page_130.QC.jpg
fa289360fc4e42f7fbf5fbc89b4d5824
83359d64a485bdb73ed4a8f81619c247a0b96815
1051956 F20110218_AACNJC franco_c_Page_024.jp2
0c95536a3ab83c4257f707c529a63a1e
9d43ffc42b4ae599badc47ce9f164e5e962448d0
F20110218_AACNIO franco_c_Page_007.jp2
c513ee8ef818d4beff3be2add7fa5f2e
45966cef8cd2a1a4da9f6ae339b41afa57545b65
1051965 F20110218_AACNJD franco_c_Page_025.jp2
86a156e92658a9f197603bc3eccf87cf
66772f72f614993d75a85fab1f6e2583d7deb2b2
682822 F20110218_AACNIP franco_c_Page_008.jp2
768d2eb1e62060b4a771b47c4e488917
399e405709815c5d6eb3a66530584d1969afdfac
1051894 F20110218_AACNJE franco_c_Page_026.jp2
57950d055882895d0dbb7ef7fe68ff1b
965b9751eea12241abc3909eba929f386b06e051
1051899 F20110218_AACNIQ franco_c_Page_009.jp2
8476063cf5a3869680e8c9da45579bf4
9991e1face0b181807bbe823f5ab4adaa770dbe0
1051980 F20110218_AACNJF franco_c_Page_027.jp2
2d24aae60fc48beb25d9c8f14415d5e1
f29de4a8256f9638752eb13e02fd5b53c33562be
391009 F20110218_AACNIR franco_c_Page_010.jp2
7e4c9980124726a767a3e0e0a3f84d6f
e2ce526e2a298ba207537e454101c2e82e29d607
1051970 F20110218_AACNJG franco_c_Page_028.jp2
1ce824251f11120a5edf1111648726fb
2fce53f8055a8e2dfb1e3d99c79e81cc9c9d5669
790745 F20110218_AACNIS franco_c_Page_011.jp2
cda8c1e85f8521c80e6bb79a30994bb7
a87959af9d12226efba27567e0a0bd7423343963
1051985 F20110218_AACNJH franco_c_Page_029.jp2
36384c92baf0641edf0b46e256b9f718
dea5e3af6c95b6657e5f77cbd61e69fee06c36ea
625126 F20110218_AACNIT franco_c_Page_012.jp2
20da3bbe044a299546164ea97d1d75ab
80ff5e5ae9294d417a74af240ced4f3765c23e4f
1051950 F20110218_AACNJI franco_c_Page_030.jp2
7fa271c6a180cd4f28922e0ce40a18dc
7abf6936867b4749bbc50f13f9b01bc7dcf6a023
849134 F20110218_AACNIU franco_c_Page_013.jp2
d745b3d0aec61566446455db607ce5a5
bb68037b8024abeecffbfcd57a0bb77340b3dfaa
1051960 F20110218_AACNJJ franco_c_Page_031.jp2
5a45a141ed3b4248c39fd59884483747
a5e8d90153b9dd6c900c14c1199b31570ad52786
1051940 F20110218_AACNIV franco_c_Page_014.jp2
30c5035aec03d7329b1f938151018bed
ef75cf6fb89628bc071f164e494b41fb199b5adc
1051969 F20110218_AACNJK franco_c_Page_032.jp2
212b135caf91b9719640b4932281b579
3cb0743ee2c38503704313a2b99d81218e5395f8
1024451 F20110218_AACNIW franco_c_Page_016.jp2
47478b800f50fb58a543fa535cd5caf8
0e2f5702f9b2793073b10ddc8338f697a545f1a5
1051921 F20110218_AACNJL franco_c_Page_033.jp2
612901ca258083660f095bdb5ba68829
f6993182778179db005d7ecceac82d2666b1289a
1051891 F20110218_AACNKA franco_c_Page_049.jp2
121db664c37f8fd15b355386f47462ed
be9d323aa1c0e97726e4de194b61059373f1d692
1051961 F20110218_AACNIX franco_c_Page_019.jp2
3279d863c375efb156bb59a94920533f
7d1b74de65e1d16a59a7b4ae2be435ae0d7694a7
1051945 F20110218_AACNKB franco_c_Page_051.jp2
f8761996552eac2195eba76a799892cd
316f2444eb8e109f1b72500a60dfa3bf604ad0f1
1032335 F20110218_AACNJM franco_c_Page_034.jp2
cfdb873ebe4c43044de0c71eb71e8397
afdd4a275e3c4fec06ddcede4afa2782bd0148e1
1051954 F20110218_AACNIY franco_c_Page_020.jp2
7465edf9537535c4064fb50f93a90b2b
d9c2811849433ff971fc9d9e81965445cd0429e5
2054 F20110218_AACMHA franco_c_Page_023.txt
b33425b8613ba9d5dbd0f76f6a83e48f
1af78501c71365a7b62bec82208ca47f8f655e69
F20110218_AACNKC franco_c_Page_052.jp2
8cba864b1fcaf8597d200160daaf60d4
fc051471df7870f7cbbd5708ec39726c38226e57
1051943 F20110218_AACNJN franco_c_Page_035.jp2
dbe17326dce842cfd932c92cc003efed
a8e07b1a0c8ef897b0219c9c9089e7bd0808e25b
1051966 F20110218_AACNIZ franco_c_Page_021.jp2
ca11ceded9159363626c1be8d0157bf6
11148afff3886bfca552914707d2faf5d374743f
29867 F20110218_AACMHB franco_c_Page_120.QC.jpg
9b2a1bb4d8e881108151a2db4464b244
a5b3a2e014e4f0e7d70dd8d417ed0ea3945344c1
1051973 F20110218_AACNKD franco_c_Page_053.jp2
1ddd508ecf762c91f630987cd2a0c879
c6583c96b9cefd1c6faf38fdb4489dbc9ff21807
1051979 F20110218_AACNJO franco_c_Page_036.jp2
10dea2430ebd57087540748bd3d0deb8
05db42a8753c4717139c5ebb214a5916c996a403
F20110218_AACMHC franco_c_Page_064.tif
14c494fdf1adc1a4e79f1d0837198b7e
e27952846507c0c50eebf4639f76dfce3044f98c
1044339 F20110218_AACNKE franco_c_Page_054.jp2
715a7ed971e0608bf7453e87958e2ad2
b9efef866900b7a1757396ccf72bfb0318fd3b8c
F20110218_AACNJP franco_c_Page_038.jp2
49ea70f66b76819e1eba6cd70dea00b5
0c070d432910b44e33c2da21c187bf2bd575b450
24395 F20110218_AACMHD franco_c_Page_015.QC.jpg
a84dd3896f2b9882bb1fc4a517fabe0f
5b4083ee4a58f430ab8609ca06882d0c87746a99
1051926 F20110218_AACNKF franco_c_Page_055.jp2
a982473591910a5f94357fa2aab26f49
c5b79d25f164d45ea119452d7e5317845e44b64f
1046887 F20110218_AACNJQ franco_c_Page_039.jp2
d0a14cc2a846f69d362ab67140f08100
4d3faf9fcaff8937781a165bfb6b2328687ea4b3
29337 F20110218_AACMHE franco_c_Page_116.QC.jpg
796c5f0efb05dd01435ec01b1403802a
bb41d905737d8068fac9e817d003b1fcd93ce151
F20110218_AACNKG franco_c_Page_056.jp2
8547f612a67643bb8e65887b2ed384e6
e1165e88e2dfe2f065152c617ff5db865aef58b7
1051949 F20110218_AACNJR franco_c_Page_040.jp2
607dc9a1856b18ffa24f7d7a497c9fad
52b2febfe9b1ba5d5b37c25ed7ea34bb59c46f33
F20110218_AACMHF franco_c_Page_052.tif
6084967e6db1034f8962c437d06f51ff
2c4d25aa074d0f3610ee917e7252fa148c840df0
F20110218_AACNKH franco_c_Page_057.jp2
4072151f3a22ffdddd104806ac639fa3
b2df7b18f830d857c8e39535c709bcd48f5386d4
1051964 F20110218_AACNJS franco_c_Page_041.jp2
f8bb5ace1ac28734e11305a753103e31
50b729653f0750fe77e7cd1c5cd872377eac083c
1336 F20110218_AACMHG franco_c_Page_102thm.jpg
5b357c52faed88e5bb476db2b304a23a
7176ca9a00ce9e18d6898d8049d11c636e55d22b
F20110218_AACNKI franco_c_Page_058.jp2
22b1ac42301d995746f8de44246d4faf
664f99f64726c62c659fe2042e9bcb0ab9d91d8d
1051983 F20110218_AACNJT franco_c_Page_042.jp2
0240e5d437eca3dba69c20c1dc1b5d55
83416941b1ec96a71631d840a9b91d05399c8157
53026 F20110218_AACMHH franco_c_Page_055.pro
7d4165d4e45161e13f13bab50f1c7fd6
766cdbc8141811a202ed4c266e1d425abbe302a4
1051929 F20110218_AACNKJ franco_c_Page_059.jp2
96c15ea76d422b761ac460b2e64bcbb2
65625cf48363c5d102fdc5db4fa77c711e8c2439
F20110218_AACNJU franco_c_Page_043.jp2
21ca5de36ddf98632b9b0c0cd8f817a4
28db1ae2ef9fca1970785b4aba1a4bed88c8cea4
29030 F20110218_AACMHI franco_c_Page_110.QC.jpg
41a6643cfec25fca188223db3a89932a
cb3a11cd517a2970bb947831f49809370791605c
1051986 F20110218_AACNKK franco_c_Page_060.jp2
449cfd156238c542abb7ebc50a8ec0e2
37f2ccae92e1df2a499c5545a187c084d1244632
718886 F20110218_AACNJV franco_c_Page_044.jp2
c74b1d95f610c96f7dec6c11454b17fb
93641096b61a5793d6fcbd3226c15f29a8883a0e
6258 F20110218_AACMHJ franco_c_Page_092thm.jpg
f1417e5ada606cd2c202bfdd0d53457b
6cf94a10cf541e38f2ab48cbcd99ee22a3b72cc9
907379 F20110218_AACNKL franco_c_Page_062.jp2
708bf437973bc50ee87479f22435bb10
27ac15cd5dea40564a7820959662981e655f2c0c
508953 F20110218_AACNJW franco_c_Page_045.jp2
f173b3d169c6faae18e80c963b1405bb
bebfac3d0e281e44d27609317a3cfd4740c8ae9b
79736 F20110218_AACMHK franco_c_Page_084.jpg
31ad9a2a36ee8b4fb3e8a5940160a74c
11d8194c0f38600269d676eb61c96bef896fd88d
489363 F20110218_AACNKM franco_c_Page_063.jp2
41d5115d1c7da19fc1085ee34307ebea
a3a42cde925fc653c0355228a495f123aebcf7df
973693 F20110218_AACNJX franco_c_Page_046.jp2
d5373e398b3f4f0ee9a84bd841296fff
63528aa355cbc183f6fee640aad4456e23d503ba
121568 F20110218_AACNLA franco_c_Page_079.jp2
763fda3b3615a6e3e37f9aea4650a50b
7539f04cb05ddda16b38937559c506a6279c9186
87553 F20110218_AACMGW franco_c_Page_078.jpg
5bed5e6ab0546389a9d9666cc84e395b
fc0bee788e9591b82fe42fe7439f31cf40658364
1051932 F20110218_AACNJY franco_c_Page_047.jp2
21187f2bb7f4afdc801132e1b73a62ae
e9217262a4cd758e754a39f5cf9e33849058df46
708210 F20110218_AACNLB franco_c_Page_082.jp2
01ca373719832773797c4ab3d206756f
0b3a7e9518606c359f92ebfc9c1f6de7dcf397c9
424746 F20110218_AACNLC franco_c_Page_083.jp2
2f904a2d21c473200b63d4003c3227d4
129401dd2536e85dc96d0ddeeed39b9f0080fc0b
59295 F20110218_AACMHL franco_c_Page_114.pro
7243ab54f43dedcf5ff6bf53096f5edb
0eae5108abf162d0c6aefde1a84071fc6afcf6d2
F20110218_AACNKN franco_c_Page_064.jp2
61fca725142be7c7622e41f30980cc6e
33f6ebfc747c012f16fb397eba91145185749383
83424 F20110218_AACMGX franco_c_Page_092.jpg
1d66ddb31a76d7dfb27dc1d34f570a89
2c3300b46f778638af5083095ec0ae33d3a92a9d
1051936 F20110218_AACNJZ franco_c_Page_048.jp2
9a5bc956ba6fc461c8ba32449827cd37
f0a2f442f7f2d72b851bd2f739f0527637a455a2
3605 F20110218_AACMIA franco_c_Page_006thm.jpg
8cdfcf825a5c909b1d0d6ac60c10125b
945554082b4ddd0b93bca0e7da128fd54c35e727
1028982 F20110218_AACNLD franco_c_Page_084.jp2
0611665b7efe8c98552c548c8d2c55f6
667b4ff9514dd592fbe61badb54d40252e7f3695
5986 F20110218_AACMHM franco_c_Page_099thm.jpg
9afbeebfae3f33537525a7831a75e58b
dae1c17a579d88878f6cf7c74899b4380489479a
F20110218_AACNKO franco_c_Page_065.jp2
8630f9e19845207453b01e7fb79b4d5e
09b18f3d13d486a716f2ab141e72f10e8da02c7b
1124 F20110218_AACMGY franco_c_Page_012.txt
dd9074a5a554e638acc6b5edb4f67abe
261edd0a15998e9493b2dea087a97ba699bb0070
F20110218_AACMIB franco_c_Page_015.jp2
16d0211afee3fee64d343bef3ef7c3e4
edcf0fe062d414cecf93d5e2c185106eaaf61353
F20110218_AACNLE franco_c_Page_086.jp2
9d1724da51e6d36f3a245ae8540ee5c7
1c1d4d008eee1bb21c6de17b9ed609c31dab588b
29495 F20110218_AACMHN franco_c_Page_121.QC.jpg
e79d91e7b0b18cf307e34fe2128801ff
7f1d0c6943fee124129c24caff600b59ad25886e
1051938 F20110218_AACNKP franco_c_Page_066.jp2
8de525e9816a56fadd38815c59e89ed4
691790bccf3179b05d444f3a614dd6cee9ea4563
F20110218_AACMGZ franco_c_Page_094.tif
a090f5cc7096fb645f2e24cbf96fec30
3b6ea956cf65b35038fa8b0ef53c27f245fc3295
24263 F20110218_AACMIC franco_c_Page_039.QC.jpg
a5ffa64e65fd34bb12ee8e1b51b6dee9
cfc531e2b298de00bf8646418dff93327fbcf6c4
F20110218_AACNLF franco_c_Page_087.jp2
4ed98a6c058a8a487fa62fb6fbfedf5c
19e8dcc9e57179c9faef8ba0009722833559c1a9
61224 F20110218_AACMHO franco_c_Page_044.jpg
893bbee42595c3f6b479be28a4e835a0
8e8c62c417cac0c3ede5ebec43ad2dedca46b6e8
1051968 F20110218_AACNKQ franco_c_Page_068.jp2
67079d77408f60795787d12ba6395754
faaf061dd5c0ce3a5b8e135c99a96c67f9e06582
59666 F20110218_AACMID franco_c_Page_113.pro
6c3478a2c576cc0060a1fbbf5c6e63a2
03fed03cc60a187c04bb06c71284daad61802bbc
1051944 F20110218_AACNLG franco_c_Page_088.jp2
4148eac831437f61e5b0cb38a1cdee87
77088ea92b03e0f50a78c4baeb565d898313fff0
2485 F20110218_AACMHP franco_c_Page_119.txt
3cca38d69c184c0baa30d420bf1ca265
fe4c0014fb1b0bc4f54db43b565305308e0c150c
F20110218_AACNKR franco_c_Page_069.jp2
6f3bed268f59e39001c4220cb0cbf5a2
d682a25aaa7f8bb9227ab67e7415711e7589010a
52504 F20110218_AACMIE franco_c_Page_058.pro
612accaf7a1e88308f23f25d1eafec91
f5d99dcfae19c1c61a593f93e9eeb63e8025f4c1
1051927 F20110218_AACNLH franco_c_Page_089.jp2
1921ec66d36c40a4b30590a29d31ff0b
8ed6d685a998ad39b4cb5fc640d8db6c61a4ed9e
1051928 F20110218_AACMHQ franco_c_Page_095.jp2
62407f4879c190b663ff46664de8de72
86d1fe1c7747b68295be6b280b5c09e56d4640af
F20110218_AACNKS franco_c_Page_071.jp2
45823630ec439e003e6c128894248a92
4bf4c8bd467cba0452618d9eea64cd4bfd7d04f6
F20110218_AACMIF franco_c_Page_107.tif
c15b9047ebcacf1b273f4e904fe71cfb
47a88d25d83820c2709e26b66defbb3fc428ad67
1051905 F20110218_AACNLI franco_c_Page_090.jp2
deb7c1396c1e9e38c565c7890181e7ef
3df9e226273253a1679701df8604c051f8ea9c36
66468 F20110218_AACMHR franco_c_Page_105.pro
0c7653759f3f9ab5d2dcd74e99ab0427
d1b0eeb5ca2b33b6c0e261b9067c66bace8f360c
964791 F20110218_AACNKT franco_c_Page_072.jp2
4412160c317aaaee69f7034d1b919b11
1814ea255a1028246bd69173ca2d8d7460b4801b
1018469 F20110218_AACMIG franco_c_Page_018.jp2
4c0b64889084699f50c35e048637bedd
b15a7c699372c83f935f7788ea3a79d15f821e7f
1051974 F20110218_AACNLJ franco_c_Page_092.jp2
1bbeac130ddb3d851fcf5a0804d2f08a
3aa439c49ea64b451dd28c1314d45fae7bde55c4
109587 F20110218_AACMHS franco_c_Page_116.jpg
957a9a56ed0c524d4dc3f9bc8e8256a1
0709e59a0511c76c5a456733eb2e03f8d45dab89
F20110218_AACNKU franco_c_Page_073.jp2
9bbe36f35a1334bb4e2e1fd8fb5b72d8
7e6a25e37377c1ef901eb31ae76a77f51aebfdd6
F20110218_AACMIH franco_c_Page_050.jp2
9e4ef7cf44bd073a76a60d3615123f93
419f6436a6a6a593044fcd4464a45a844d561114
746619 F20110218_AACNLK franco_c_Page_093.jp2
8c80a2dd88987351df9d8a0c9706f737
c2ad366b13f52fdd61fc5fd0a0c135496b99c68e
6347 F20110218_AACMHT franco_c_Page_069thm.jpg
708b7342ea76f7c1f20fa4ecd416af3a
6f5159a5ee82a626f4813abbbfeba3f8f2bab8e3
1051981 F20110218_AACNKV franco_c_Page_074.jp2
8d7ba310405494820856cc68a71c7d27
926f5996bcf843ca8a05ae0f8a087d36ce0f873c
515396 F20110218_AACMII franco_c.pdf
a6284a5b50c721274ba6553781591fd8
be750b779aa9782eab827c536807665d7ea42ae8
F20110218_AACNLL franco_c_Page_094.jp2
c7f1d5a88437fe6558bfe329f4d1901d
c6ac877cdb248007d9c295c05da2d2d873683b33
26145 F20110218_AACMHU franco_c_Page_075.QC.jpg
a2269f053408f322280247456582c030
f6eab48802d82eb9424260ba107d84eb8072bcaa
1051934 F20110218_AACNKW franco_c_Page_075.jp2
365539c5d025e86e0c176189ed77f077
1b0f014a884a889fb978da7aeedea1fd13db9f2f
5794 F20110218_AACMIJ franco_c_Page_028thm.jpg
4ab7fa23f7d020942a45edb857521213
3b21f02bf140db353427645f4f4e5f06bdf9e7d9
F20110218_AACNMA franco_c_Page_111.jp2
1aff9bf38f79b8020069ee038aa1badf
1c621006fad0ac232d1d496332335b116ecd9b9c
F20110218_AACNLM franco_c_Page_096.jp2
3dc637aeb0e01ecdb62bcf18851ee7a8
7d2c62cc97f1653a429b0ac01aab073bd3a3e18e
104020 F20110218_AACMHV franco_c_Page_131.jpg
ae07a2c5a6fa3d1cab9e62ae2dca0689
508ffe66a50e4f96604c65552ec33b88e211836d
1051911 F20110218_AACNKX franco_c_Page_076.jp2
9501ddf46824b09a59dd599def929cd9
8a33e1921ca1d5c157f2b477cda6f19aab88841c
58738 F20110218_AACMIK franco_c_Page_130.pro
f483a64016d3d61f2a7d46568013fb79
0934af4e4d556c284167c93e0d30b304c1472c75
F20110218_AACNMB franco_c_Page_112.jp2
9adae4cabd459be1c1f17a6dba417c42
4f3769cd6ddf1b81dadd94926c8072181f4a1424
1038933 F20110218_AACNLN franco_c_Page_097.jp2
302c284e273e51b9028ea9efd524fc64
6991f5b8804df0d46eea72c969d1dcfd1894172f
6500 F20110218_AACMHW franco_c_Page_050thm.jpg
3ba1fbfde4f2d8a802662b3ec147c9da
d83bcdb521c647928f27552777a2069a4fd16717
F20110218_AACNKY franco_c_Page_077.jp2
7e1cd9f9b52afeee4fff896e12ae25cb
7e686a316021ef516c84cfb7d1bb8931f3c60927
14770 F20110218_AACMIL franco_c_Page_045.pro
3da7f14db449edc3702e23e4b70170b9
bfa87963e951fe3c7a9ae813d5fa1778f2ff3abe
F20110218_AACNMC franco_c_Page_113.jp2
515d60f28f21e3de91d846705745b6a3
04f372862efe6ed95b8a6924ed28e62333576149
25336 F20110218_AACMHX franco_c_Page_098.QC.jpg
5958cd97d5fc86d82473dd7f77f3e4ec
a11be2dbe981fbb2e60ebec9da9de96c4023d8e9
1051904 F20110218_AACNKZ franco_c_Page_078.jp2
9a3645f5fb75d7c0ab7b1ca641ad3565
2e1be8f4b76f7902f17200da6108844d8609b8c2
2521 F20110218_AACMJA franco_c_Page_122.txt
be71dd8084d1dde69c26025301a901d5
4d62b0b58483cde8fd9a42027ce2ba69a1da6a8b
1051984 F20110218_AACNMD franco_c_Page_114.jp2
a941a229d006a891e6ec7f7b582daa3e
4d303f34a64146fb741b98bb391f30d654a8cb76
F20110218_AACNLO franco_c_Page_098.jp2
ee0b37d377e274829a64f267cc7161b1
fd3c0b6b6adea5d24b9afde9cb4fe72c9ed9a1c7
1965 F20110218_AACMHY franco_c_Page_066.txt
40745b192420929c88bed4bf6732348b
11aeb2ba7e0578f502a484804283498c6178095e
20467 F20110218_AACMJB franco_c_Page_009.QC.jpg
fdc869c60a49e64ff00606f53887f6ac
c39f7b033408ce9a9402888800306f8815381dd6
27999 F20110218_AACMIM franco_c_Page_125.QC.jpg
b8e14706cc000ea040c37cc95ef6e81d
68fc8d6d8638f223ef1b054734bacae00d0a0081
1051982 F20110218_AACNME franco_c_Page_115.jp2
80fa64910ea14d3247d6204fce21226c
b356cf374aa493999c767a90dbc22af3451f711e
1051933 F20110218_AACNLP franco_c_Page_099.jp2
ecb640f99faba3836c329f61da82f2f0
645b47535022f77b53f9d6df34257c3895882ba6
28613 F20110218_AACMHZ franco_c_Page_111.QC.jpg
96cc7d183d48425d93f51f9c967c2bbe
bf6dc0f88bfc102e862970351ceed3bbe77d4ae5
50365 F20110218_AACMJC franco_c_Page_077.pro
eb019f1e16374bcf194a2450dc2afa7a
a4039f26b3f1daef4fcacd3beb60e4fce7b1ed73
24719 F20110218_AACMIN franco_c_Page_100.QC.jpg
5c027d6bde0c2ddac8ad225e2f7703dc
78e2ecd4b688c0373e261029f4e3c27665a61cae
F20110218_AACNMF franco_c_Page_116.jp2
837ecbe8e4b84a5ba42660b39573f8fe
f0eda0205cd542c1a4148c261ffd97422a7480c8
F20110218_AACNLQ franco_c_Page_100.jp2
1ce01410beddc7b81ca457b077026106
82f8d220f54d5a9b146177dd61eb8c9f91f73ec5
F20110218_AACMJD franco_c_Page_040.txt
72651a71ac2ad80becc7776bd2947fcb
1b16d843e6bd8c3a0096dda2d198d6df130788ce
F20110218_AACMIO franco_c_Page_074.tif
ecdf00446b600c5ce2d3f635a9b7b289
0663eaabe8301b9e466b982873f645ba3869e566
F20110218_AACNMG franco_c_Page_117.jp2
09e485931f1291b52cb1ed98fa2c95b2
f26c8b3b42f09c5bc112ba00381ab0aab952e646
167026 F20110218_AACNLR franco_c_Page_102.jp2
480542b661f3b4f5531baba5c0eb4ec4
949defc644a02e913ab96d6c81de6b156f829ab1
2027 F20110218_AACMJE franco_c_Page_070.txt
4c5e33ba863490d3a79cdd1bd418192e
7b89097ce0bd6438bcccc73197fcad357d77e6d7
2048 F20110218_AACMIP franco_c_Page_075.txt
2675b21694cae8de260e221060b13bae
c2d29594f980d777c20085a79b76123ebc2757c8
1051920 F20110218_AACNMH franco_c_Page_118.jp2
54a894c7dd6c59a08089770f1dce4f69
9405889472c903fa15aef27f0dfa59e75861fec9
1051959 F20110218_AACNLS franco_c_Page_103.jp2
f44f2cacc86fb92d8b33e7e28ea7f947
cdd40c3f8f668b9bb5e56fa1aabf776458d74ca6
50831 F20110218_AACMJF franco_c_Page_087.pro
52a9055ab5adc49eba99244a472ce6d0
003a3eb308553267ca24ad39ba9850de7187178f
45883 F20110218_AACMIQ franco_c_Page_005.pro
5c4cc747ea7831d157c77168d6b9cf76
7e3c6c34a0e83a8896ec7b670eb5cbf2c85e5d48
1051948 F20110218_AACNMI franco_c_Page_119.jp2
144e93d7a999776525c51b38cae2fba7
e5b9a3c769b45ea8555c46d5e3f53e36fceea2ca
F20110218_AACNLT franco_c_Page_104.jp2
7999b4760b8445e14ebbe1fb389100f2
c9a3f08fe1c484e386756daa2ba09075c3960910
51256 F20110218_AACMJG franco_c_Page_065.pro
a44709a7ed500fd35a36f72eb451a3a5
55c03b958d0c4cea696220cf028229565af9c4c9
82464 F20110218_AACMIR franco_c_Page_033.jpg
5f5ebd036d73104ce8bc65e90d02d176
3739f2f030fcbbe9ff5a08d7e94faef02268429a
F20110218_AACNMJ franco_c_Page_120.jp2
a58afbbddefeafc86ebeebee007e4e26
ead68b314820cd4bfa676c49352e49feabfc1994
F20110218_AACNLU franco_c_Page_105.jp2
e1e0598b4f2f3eb5a69c0b7999d3f597
774fd846cc13bfee8a742a109e107b2b48dcdce1
673042 F20110218_AACMJH franco_c_Page_081.jp2
5e6724dab81930dde3819566310837a2
53d9c32fc2cfa8581f7074db5f9bfa1113532714
6545 F20110218_AACMIS franco_c_Page_026thm.jpg
6e5e4c103ff119c93eb2db2ba0ea6469
160627820a8d4c5663f8503c84e59aa227e9b2bb
1051978 F20110218_AACNMK franco_c_Page_121.jp2
4fb076ee080ef36f7689509dbc1a3279
cd9c1812c1766a91f6581d7c41ebc8262143e80e
F20110218_AACNLV franco_c_Page_106.jp2
4eab84e16094172dea647be18b041371
8192829a23e1f8c03f18680d793ab2042cf91656
6003 F20110218_AACMJI franco_c_Page_100thm.jpg
821c01ace4b0a2abd91421ceffe365d2
b7c7bf77c518b5f885a76dfe0f32aef3a57e827e
53502 F20110218_AACMIT franco_c_Page_026.pro
aeae23a996b0b2dce2ae5bd5ec6c46c2
74f0c10563072982adeb509781ccba1e1057f2ec
1051941 F20110218_AACNML franco_c_Page_122.jp2
11004acd2139cbc8221cef6ef732d293
fb58f82c76b21b992a9917692ddf50b5facbd7af
F20110218_AACNLW franco_c_Page_107.jp2
7c3f9567c35493e6840524d603adb162
3eb2c8546d95cf56bc7e714eafb8049b1c2550e6
F20110218_AACMJJ franco_c_Page_083.tif
f15a631faa00583bea562958ad2aae87
270c8bbca8ec899ac81493507789aad8feeca67c
F20110218_AACMIU franco_c_Page_076.tif
ea2685964c14ea1e712d0ed06adf57a2
62811928a37320b7729f528454b390a1627b6cf7
5984 F20110218_AACNNA franco_c_Page_005thm.jpg
1d4f156c639a7413dbc09ec4c6fe7ca4
2114f2fce5d1da0f751a6f392a00e5dfb788eaa7
F20110218_AACNMM franco_c_Page_123.jp2
5a9a99b4c6fa708432e70c78029890c0
f35dceaf2e2aec9d9d36edba742899e5c94553f4
F20110218_AACNLX franco_c_Page_108.jp2
e78d81e272eb005af6f9f2fc5069b463
e3bb7c065c0af0b494c3b174258e5f0a2f918f41
1051918 F20110218_AACMJK franco_c_Page_085.jp2
93802e6c1665ce9d8c68e3d102b7d923
c36b3e61e25f2c17a9ba373244663f1159975780
1051953 F20110218_AACMIV franco_c_Page_130.jp2
617878670d22f166ca99997b65ab128d
2f7eb3a41c8f6cb5519488c5b77548a1205988b1
5670 F20110218_AACNNB franco_c_Page_007thm.jpg
d6290deda76b546bf90dcddefcaf8396
7a7e618c2955f06a94845565d5d9aa198e70b4b6
F20110218_AACNMN franco_c_Page_124.jp2
c86c400fd3a2aa23abdedd1585d3fa8c
80b9828de9ceebc0bf3d6697f5ee017478077183
1051972 F20110218_AACNLY franco_c_Page_109.jp2
2a75d215f78bfd3538504b089cabb5d2
ddca338a312f30c61bec3b6cbcf29905c515c075
25622 F20110218_AACMJL franco_c_Page_020.QC.jpg
8a30df365c1cc4871bff48d15415841f
f3b2b09ef0fe3660a4b82f806887cc64e0ca26ca
61697 F20110218_AACMIW franco_c_Page_118.pro
469e004a5d38e5046d410eaa19b35633
7c0da5970ac82faf94b355a2d6a129a47f5c4156
2906 F20110218_AACNNC franco_c_Page_008thm.jpg
5f1c2369b05c941afcf08541e909b71d
a4e1ecbbf72dd18eae31b1745eb78483cb1f37d9
1051977 F20110218_AACNMO franco_c_Page_125.jp2
5bede8827ec0a5904770f6dad87fc844
0315e8322c47d4a5637c3a19d66a4185c7732805
F20110218_AACNLZ franco_c_Page_110.jp2
0a4d03f4ef39ebd1c1f82159200b8379
539c54c17015bc19f0b6beb8b2dc44bed2a1d140
60493 F20110218_AACMKA franco_c_Page_125.pro
00d781593108ce1c03e18c26408500a4
d31e52c00e5d28264ab742ca2ec1d01e4ee0e37d
2435 F20110218_AACMJM franco_c_Page_114.txt
1c7dea3cf9c584fe8f4acbe48e67cef6
58395a7c21e8905f3ad1364cbc5877c92a08dbad
6114 F20110218_AACMIX franco_c_Page_103thm.jpg
80aa063e54fb221b955feab0da15984b
f38f918e374a6a41827e398e3c8d5f0bc22b4964
4626 F20110218_AACNND franco_c_Page_009thm.jpg
b57bc79f5b356cb4669f5e11ba06f0fb
fa20de66bf27056e55cfb26735c19cf0756a6199
F20110218_AACMKB franco_c_Page_070.jp2
6141f7cc934293d7e127d716fe8ad351
b9cdef1902449f47b690cd77de43d84f862c5e09
6263 F20110218_AACMIY franco_c_Page_066thm.jpg
d56291fa272d5174c18c279c932e17e1
3caf41310203ff7f05767caa32fdb8a3442fd1d9
4500 F20110218_AACNNE franco_c_Page_011thm.jpg
1877b1f32ae49c9368d1737e42639a6e
786907d01f5d427f67207c48f1d52aa4cf64d0da
1051947 F20110218_AACNMP franco_c_Page_126.jp2
0ac74c02eb047875a97caac1a318f0e3
e56e90a4587124bec48fef758e20656e7394ac50
48292 F20110218_AACMKC franco_c_Page_100.pro
a3d06f61d06776e4644960b8f51a9276
0093ac7535d851111604d0d27f0fe92591baa68e
5683 F20110218_AACMJN franco_c_Page_018thm.jpg
f4f394ff4810f2ae581210319131718e
b61599b50dc3a48245688332c23715792c275db0
6328 F20110218_AACMIZ franco_c_Page_021thm.jpg
14b20e1b44bda06bb04e631c1c9ec89b
84a94c0af0d87ad4cff08d14f2e85bcd3b6c15c5
3597 F20110218_AACNNF franco_c_Page_012thm.jpg
081a70c5191e7da25e29cae3dab91ba2
3361c20428dc3ebb9365d847fc8a88fa0b060093
F20110218_AACNMQ franco_c_Page_127.jp2
1c2dfc833a2d76958f2ee033e1087794
9f50f6383ffc0b8f15d1274d2fb573f9479482b4
64029 F20110218_AACMKD franco_c_Page_117.pro
b54f3bfacc5666999291e5106d6438da
1c362984f88d93f537fface26aa2c1ee8d19528e
5350 F20110218_AACMJO franco_c_Page_004thm.jpg
448c39a655f4c96803eb3a89a56d30f9
bfe147e994ca2dde24459098be0a7cc158624dc1
4846 F20110218_AACNNG franco_c_Page_013thm.jpg
b325a033beec4137558dd9286be93f3f
484bc439151ee72bc901b7f2689a1f6574dd4a52
1051935 F20110218_AACNMR franco_c_Page_128.jp2
d750b3ebf937cfae99ebc6111096ad73
fb91cb0a90991db8757f01309c2a6a9407c4dd89
F20110218_AACMKE franco_c_Page_054.tif
42ce978b38ea8b8f50ea12eb8d6a7fcb
cbae2d3a12158a9c3561f76e706e3ee7acc09028
6090 F20110218_AACMJP franco_c_Page_101thm.jpg
882923d8192c9ff196025a575a34834c
3274efdaf67f5cc7c0cc723aee4a59e44b5d97d5
6078 F20110218_AACNNH franco_c_Page_014thm.jpg
242c24415c4faa5991e84b2295312224
832bbf6729fc372071094e874c1d645fbfe8c3a4
1051962 F20110218_AACNMS franco_c_Page_129.jp2
2aac89afc9a9448e9a0ea1fc1e653b5e
7952bdc980b15013c79347cae9f38ea55f1aa5bb
67773 F20110218_AACMKF franco_c_Page_133.pro
61060506aa35fb06e6dc25493a54c7a4
bd53895ab2de3e5e238b3adf5d568743ed42c540
2382 F20110218_AACMJQ franco_c_Page_130.txt
3d8f8ab76068193703853b51cba3b4f0
02df0b8c3f67333731f8a9d2908ed68f2ec0e2d4
5947 F20110218_AACNNI franco_c_Page_015thm.jpg
75a26d8b2abb4764b8e6ab14ffd8cb8e
9a94804697ac3aaa45cbf7d33b5b4420791b3581
F20110218_AACNMT franco_c_Page_131.jp2
72fe9e9f36f9a0027feae9f618eb5f46
e4cfda565dc9cdb98ecd86c381bd6882194ba2be
1844 F20110218_AACMKG franco_c_Page_039.txt
2cac7838ea0758d627d41d6bcaf7b875
7edf40dba8c51e14fde6bf08da3d94cec9baa4ba
6300 F20110218_AACMJR franco_c_Page_042thm.jpg
e01b80b56ed396ae31e4d9e54bc05d03
04fff67ab21c752ed515acb98a6762f313429295
5916 F20110218_AACNNJ franco_c_Page_016thm.jpg
9a03d618a182e35f5437f4c5e9b3d293
13eed79ad338187ca2b5e56ba327076d5210e4ef
F20110218_AACNMU franco_c_Page_132.jp2
d162c3b08e1734d75efd3659f1dc45dd
e58cf0a568bfd93982cfeba8bdaf2b7706f2d102
F20110218_AACMKH franco_c_Page_079.tif
98d33397efc3cb45f2d78b4e880fd751
16f2520954a6233866f644311da41bf08e909d75
18495 F20110218_AACMJS franco_c_Page_061.QC.jpg
6c677d32d3ce498bcad918810a0e1a77
73584d573903471f3114be9bb3be7d914ab1283c
5949 F20110218_AACNNK franco_c_Page_017thm.jpg
7ef008532cd7dce3e2a3d508d579e092
b28fdd3b8dcdd40fe41615105dac7394c74b4f6d
F20110218_AACNMV franco_c_Page_134.jp2
8f143c946b790c77128c8877eea64dc0
f57b13dc53a328120e5d6ce7c21febec61ba7c03
F20110218_AACMKI franco_c_Page_101.jp2
3f606bc92112400439553c7938d771da
b3be23b5f17a98faba7e45d85b10f86bd755befc
83476 F20110218_AACMJT franco_c_Page_052.jpg
16aa9e3def2aa946151bdbcce9441040
8a7b2258710beec91b2bb6f0f0d9238a8cf33263
6181 F20110218_AACNNL franco_c_Page_019thm.jpg
3d38d6730be2c6b9093660cc9d64da4b
88a7275248fa68ac8bcc28892853bee0d17464da
687269 F20110218_AACNMW franco_c_Page_135.jp2
b01c4bb28b81d16d0a7b43c65791545f
5bd529447e7dfbda67357365ac9eb30509722c42
29685 F20110218_AACMKJ franco_c_Page_105.QC.jpg
f5553b0b5d9121e6d2b8358884926459
49b2b1fa1c71b569a6fa2d4cdfab3f39e8de56ce
103531 F20110218_AACMJU franco_c_Page_125.jpg
edb05cb1789b6880f140247e92d2eb81
3343c5e49543a37c38c7495acec6e1240a456b34
6392 F20110218_AACNOA franco_c_Page_037thm.jpg
d92fe4c49c7e301fc5f90169da840316
91424e9f1f751019c0bacf9aad87a263fd46b0a2
6070 F20110218_AACNNM franco_c_Page_020thm.jpg
3641f3c182ca65d12a440fe2c49919c3
7361e751bb85cf6cdfb9b9b8f23422e8e1f7afbc
1746 F20110218_AACNMX franco_c_Page_001thm.jpg
3d603f861816af236910fbb2896daed7
9f57ae8f4e4bdaa9645aab887794b0dbf0399909
1051843 F20110218_AACMJV franco_c_Page_080.jp2
858d475ab1e12b59ce25439214e89f5b
8e1e653bfe368999c104c2b3180116d1597659a1
F20110218_AACMKK franco_c_Page_043.tif
93a74dc12393283c8f81b9550cbfae64
8f098bba091e4c7afc37167123612d6807f28084
6080 F20110218_AACNOB franco_c_Page_038thm.jpg
4d6c52c9424d276bc83c97a44e3d545f
2b1a2b74637bd7232787c06a507664832e88e913
5853 F20110218_AACNNN franco_c_Page_022thm.jpg
d875a6851dc2f5d27df6eb420cb6fe0b
9721c5fbdec6c5dd97f9f978ac8b301434fd95f7
530 F20110218_AACNMY franco_c_Page_002thm.jpg
4c4ca7750674e0b35ee1c9f9eea7ce2a
9dc157aa8c4c8cfa811a6c198011c8af120656f6
2059 F20110218_AACMJW franco_c_Page_058.txt
b3562a34c7a12ec82b06c7da052bd5a0
01188e8a7e21b9de5128ca3253e8965435bffef7
2466 F20110218_AACMKL franco_c_Page_115.txt
10c673933834cb75d19c616c9b26dcbf
39f098652bd7ec16953c98043260ad101a4e11a4
5951 F20110218_AACNOC franco_c_Page_039thm.jpg
2a08060a501814b6fb1c3ae94158cb20
8884d340c7317889e4ae85f0bc3b3b90263a4ff4
6218 F20110218_AACNNO franco_c_Page_023thm.jpg
25077ecc8934e330e275ab92ee411e4e
b99bc8be9ae5e69b7c485a4dfb5e4dbdd1fd13d9
2125 F20110218_AACNMZ franco_c_Page_003thm.jpg
c86f7c11e61f4859aa21d5de19f3c6f3
2ac2ed06ab0bd43389d24f46082e46ad8b6ee755
F20110218_AACMJX franco_c_Page_088.txt
ce5c15930f527a04a512eaae7e64d8e5
4a179293a48a832e5624dbccfbf67827c2d1ca64
87888 F20110218_AACMLA franco_c_Page_031.jpg
6e6143f3ad80c29d796d9b543c643af1
2c18243e3a98081f8078435aab4b5855f7402b57
789610 F20110218_AACMKM franco_c_Page_061.jp2
8becdd25a0577f4526548113a10766cc
8f71c933520a164bb2702dbd42dc64c2d82e51d7
F20110218_AACNOD franco_c_Page_040thm.jpg
143418579af1da217ff891b1a0dce216
6168a1527746da90195f7941f2b931b86f65fefb
6158 F20110218_AACNNP franco_c_Page_024thm.jpg
cfe7d539f74d93648a2f121c668a98bd
f210ac168219fc242d9d41d9936c119ed8461a5a
F20110218_AACMJY franco_c_Page_037.jp2
5b02e4d8865b9926acf749501e75ecff
be4254b93e1af130143aea63f0a2a3584912575f
23982 F20110218_AACMLB franco_c_Page_084.QC.jpg
e0ff5b781a80731c840c9b9388c2b622
d4a88d139895d86fabf228789a2ffd3873a87d88
F20110218_AACMKN franco_c_Page_121.tif
347a63d75220ebd2f16cba588d74b3cf
0f682f48a92ac9adea17585b328c3e793805b4fe
6191 F20110218_AACNOE franco_c_Page_041thm.jpg
5cbe1b12c4016e6b5bc60e80d5a7d4ad
288c31a5fb5725ff1f1e489ba17362436049db27
76288 F20110218_AACMJZ franco_c_Page_034.jpg
9d5ff3effa24ddd58897a2eb3c261e56
69d608809b9e0fc6a031c65412f12e322153ff90
24291 F20110218_AACMLC franco_c_Page_094.QC.jpg
a22b5781714203d83aab965da2eb01d9
784b6bc78d419830668eaac2340ec3ed5423169e
6110 F20110218_AACNOF franco_c_Page_043thm.jpg
dacd039dbff3e8dcacc40ffc19915630
afa7ad04166b3a18de023ae91d57a02d9092db63
6430 F20110218_AACNNQ franco_c_Page_025thm.jpg
320e2f3cb72632b863e7aaafadd494d1
ee9fd4deea6cec4d74a7baa1b00cc91977267a7e
27702 F20110218_AACMLD franco_c_Page_113.QC.jpg
1fbf65420191cb84f21aebd3bc93c85b
9fd5847711d666e301681dc251d8d620073b684a
26511 F20110218_AACMKO franco_c_Page_088.QC.jpg
506a01236e3e48fdd058d2c720eee9ba
50534a120c96efe470b10215b44081e90280567d
5592 F20110218_AACNOG franco_c_Page_044thm.jpg
9c0ca1caaaf52f6d498912d1ef82c483
1f91c8af70d462bbe7e069589c0ef1b6ca34055c
6290 F20110218_AACNNR franco_c_Page_027thm.jpg
73539f850b86925c40aca43e1507163f
4564257d36a513f294dfce5985745e895cd4d104
50502 F20110218_AACMLE franco_c_Page_049.pro
be619297909fbd7b64c9fcad24bbefdf
52099ec5e3fcfd58bd44ada95add13dc452de99f
106031 F20110218_AACMKP franco_c_Page_130.jpg
ab03737c65e649e0354a33394d3af591
fb272e68633292ba0e2724efbdee11c05673610f
3940 F20110218_AACNOH franco_c_Page_045thm.jpg
06b6148889d8089d61209cdbdb4c5b6f
2bc7f1af3c6c58e114f8a2f444d06d44d5231fac
6404 F20110218_AACNNS franco_c_Page_029thm.jpg
4935d352bb021fa6685113560304edfa
5bae9a21f7a48a4a816b07d74d80e463a5ad6799
F20110218_AACMLF franco_c_Page_068.txt
f63c5d34671f6c401b153cfac6bb782d
4d73ff36d09cd8e2058d4ba89630e6a7e65c9a75
F20110218_AACMKQ franco_c_Page_133.jp2
4ff37d0b03aa9aeb9cc0404a90a81b3a
0c96be73376cae146fa12fece140f792db91ed00
5482 F20110218_AACNOI franco_c_Page_046thm.jpg
baf81a62bb2ce4b71de943129add3f65
07199c04c027de7970a9e9d183f15bf2fe4409ab
6075 F20110218_AACNNT franco_c_Page_030thm.jpg
87c89e64ba2830a0f33af81e826a2cc5
868560f44304f0b08b34c030d46c4985ba81763a
6159 F20110218_AACMLG franco_c_Page_065thm.jpg
9fb7fcd612e578927abee1d6c50d17db
9018c13e6af3ac801ab3da18da67b050561882d7
28242 F20110218_AACMKR franco_c_Page_071.QC.jpg
232f4d07aa1781bf084bd1769348b06f
f30737d3748e53737303253277ba385734a28262
6318 F20110218_AACNOJ franco_c_Page_047thm.jpg
e29914162d6893c8e878cf187aec4c9c
20eecceb45644d638d1458572ea925954c3e36db
6422 F20110218_AACNNU franco_c_Page_031thm.jpg
af763f68b5fb13144318e8fe3a1e1464
bc90ff7da9c9bc98d3a2b328de865b96a0563ab8
1848 F20110218_AACMLH franco_c_Page_097.txt
ff9358be21edc0cab63587e9704ca3c5
28fe28edbea3eb95ecdd49b5f1683a328857ca50
F20110218_AACMKS franco_c_Page_097.tif
aa515528a41ac0f6efe53218ddf3e66d
08f1f1030e645c4563402959184367c9ec9c9d2a
6522 F20110218_AACNOK franco_c_Page_048thm.jpg
1ff23010c6c2d441f30869c5798bcc31
29df1c1bd5c740c5acc39ac6d0b543870ff5e449
6236 F20110218_AACNNV franco_c_Page_032thm.jpg
20be6635dafac6d7a531bb0aef844a3e
17e0296c497b1105da58c204ebe8973b739f2d38
F20110218_AACMLI franco_c_Page_010thm.jpg
17ac295ea624f3865acede4956742145
ec70939f551395cbd5e44c11b693626d4f4e8fae
47142 F20110218_AACMKT franco_c_Page_056.pro
8ec840f20c401b76f7d0c12fdd947fb7
2b7f5e21bb5789ad2220217e6a081cedf6f16759
6461 F20110218_AACNOL franco_c_Page_049thm.jpg
5a10aabf19cfa241504b2bdc805c075f
8f69c8070f93be3e60cdb08c7a91801c3c8f9b95
5913 F20110218_AACNNW franco_c_Page_033thm.jpg
c17af0571b37e69fb5642b254a2f39b1
37bdfa57862229e4601e2e1480c2bb495d44ef26
F20110218_AACMLJ franco_c_Page_047.QC.jpg
35e341c38ea543fd1d9a9b49e3a790bd
8dad1d1e692006749b0af6681086b5717fd63636
81074 F20110218_AACMKU franco_c_Page_101.jpg
dd8f66b164f9c31c1778a94a94151eac
df1bd3331d3a660a3d61ccf4ae63aca4dfb76e98
6403 F20110218_AACNPA franco_c_Page_067thm.jpg
4d6189f846f51205c53b65a706025437
b393b8b607fdc556681e69880a96e4576d65aa87
6299 F20110218_AACNOM franco_c_Page_051thm.jpg
f99a569e8634f8bbeeb54b43188aaedd
15fbf156d95aaac7bdec95b91b95d77240fe5d6a
5836 F20110218_AACNNX franco_c_Page_034thm.jpg
d0f1c94ebb5fd45cfb9d565f96ebf275
aafcb241be4fbe55b91e58e58236b1fc0a7f77e3
2577 F20110218_AACMLK franco_c_Page_106.txt
bfa3bf16db45c59a1974f3056d8139c5
6eccc1f1c03ccd7aebcffba992d8880c5f0d76d2
F20110218_AACMKV franco_c_Page_100.txt
b1d7582bf2e632b466362f28127eb64e
4a68fe14f0c76e0c02725c966d756bacc879c78c
6274 F20110218_AACNPB franco_c_Page_068thm.jpg
96a8840e7f8579bdf4531a7a8494cced
5411e64b7be097dc698245e6ae1b311caaa5b3f0
6116 F20110218_AACNON franco_c_Page_052thm.jpg
35ef9702947e54bf2f1e481e4591988c
fff87d8f9a9dd3e76b776fe51cf1a3efb74d6e41
6453 F20110218_AACNNY franco_c_Page_035thm.jpg
2bcb168e3db4d3fabb935b4fdc3bd5ec
6f268605a8bee5a562a46d268577ee7f5bc47f02
110599 F20110218_AACMLL franco_c_Page_121.jpg
f509b1267d38465da98a49f33be36a32
596d621971b6b19892e80f03928d688a046d8528
338756 F20110218_AACMKW franco_c_Page_003.jp2
232b2d7c4df84427dffb0fd0fc068556
29cf6689e9be43cdce0996bba0a53cf5e405fe6f
6423 F20110218_AACNPC franco_c_Page_070thm.jpg
6e6fa4f77c0deb41fb0d222934da3026
45845998fb71cd06c1da2e3b188702d5c8459152
6173 F20110218_AACNOO franco_c_Page_053thm.jpg
ac0b9bdcdc940a01bb7b02b6f7ca7f04
31298524cac23771e53521a2e4ffd7704142cead
6431 F20110218_AACNNZ franco_c_Page_036thm.jpg
4638bc386e285c9c1dc40bda90c5f2d3
0a89efde813d338354a8d8a76e33195c7f076cd7
F20110218_AACMMA franco_c_Page_014.tif
c16471debedffb965ae886e1aa763bf2
01cc5eb21b5d69ab81c016dadb5e93304878285a
F20110218_AACMLM franco_c_Page_123.txt
45845cc1467d4bbb54038aeb4abd9631
315f17988f219ba690dbe7036587771e336e4ed5
27079 F20110218_AACMKX franco_c_Page_043.QC.jpg
985c7eb6f7c31da863ceacd93c109ada
b78b21abe5a2b8ac8672fdf2c86a62bf016b44c3
6579 F20110218_AACNPD franco_c_Page_071thm.jpg
13bc0c7543ea8fb5a2b2f7f0a11249d0
836b7cf6f439ae6b68601682a8db976f39c71aff
5822 F20110218_AACNOP franco_c_Page_054thm.jpg
94188b94d4277bd680b126ecd2244e0a
0cad01fc24d3bb37da3ca00b620cb8ab1cb8f6ad
26228 F20110218_AACMMB franco_c_Page_103.QC.jpg
a8b29624072145e0240fc3d2efaa2b2e
d027ed19bd19dff2c4d098486ad6b45a595a2403
F20110218_AACMLN franco_c_Page_002.tif
eed118c7c261068806db31fc72761282
9db9ffc326b435c4ea7ac0add1e31d4969706eeb
F20110218_AACMKY franco_c_Page_067.jp2
918b3d7d3621cc69578206cc18070daa
65acde2fb4942dbc518a553b74342577343e6d3c
5501 F20110218_AACNPE franco_c_Page_072thm.jpg
13b56ebd959ecf052c091981c0f6aa43
d70fd7cb8c95cfb1974d1f15891a5a767a379422
6625 F20110218_AACNOQ franco_c_Page_055thm.jpg
cf9124c97e31afe516bd66f8335e8b69
c50d30cd6bbcc771cbe419ea1821aa6aa98b61c4
85635 F20110218_AACMMC franco_c_Page_070.jpg
9a31a1b3a2db2a4a8d0b5935fa0bd30a
7fc80daacc1c36a4b345cf66f0e4eadd447045ba
1024128 F20110218_AACMLO franco_c_Page_017.jp2
8b9e895347a0af59f44172202be1086b
1e97f902d1e6263bc698b8145909bb8313162cb3
49051 F20110218_AACMKZ franco_c_Page_066.pro
2810d99e0ceeaf6d856deaae47599efd
9ec5d2d6206c98eaed01a11476efe9f6038838e1
6289 F20110218_AACNPF franco_c_Page_073thm.jpg
452af788b68389c70a1c28b6515a8cd4
03653377a42aaf934073144b9c4b3d13b32b8f71
47544 F20110218_AACMMD franco_c_Page_094.pro
5deb9291608d0935f81bc056d3f23ea4
6dd72e00c58dd260bb127c023362105b9be4146f
6382 F20110218_AACNPG franco_c_Page_074thm.jpg
253ede4e01be3046088446242801b8e2
e17e2597e342c4322d00be8c8d85051c247fd5fb
F20110218_AACNOR franco_c_Page_056thm.jpg
712b44c80d73f58832ff5481ca8cac48
574acdee82ea95cd2d809d436518c5be928d3348
61896 F20110218_AACMME franco_c_Page_122.pro
e80fb246455b45165f77098fce9eb2d2
dbd4321e021a175633fbf2fd04b0481746d99a7b
60193 F20110218_AACMLP franco_c_Page_109.pro
8789b3c6b35803b0790200929bb5235a
c58414fb1d811cc5a87d4b8e5584a0b800616e80
6223 F20110218_AACNPH franco_c_Page_075thm.jpg
97254a08713d1691c879f60967dc45a2
64a70932108a797b8ad3cd4044405a2394289b98
6281 F20110218_AACNOS franco_c_Page_057thm.jpg
d14671852f2849157109254cc536275c
841ec99d0300066c9649338fbd85a491595a79f1
218271 F20110218_AACMMF UFE0013409_00001.xml
9a7ea2fc8d8e13b5a738b2d9be606a32
895b1d40dca3d1cb65e3c375bdffcb7c82abd084
1771 F20110218_AACMLQ franco_c_Page_062.txt
6d5bf114baa118d882e426c1a9a23d25
81f2f60ad319db6764fd0b384cc3c2b7fe6447e5
F20110218_AACNPI franco_c_Page_076thm.jpg
e25b62c89b344461f61833d71fcab4e4
2bd38e829a693a67d520d65b4a4a5e8823b243c5
F20110218_AACNOT franco_c_Page_058thm.jpg
65a468d7604891d55a9cfc8e2ac17d84
91172e94416eeae51a790b99f0466e5ac8d52600
1998 F20110218_AACMLR franco_c_Page_049.txt
6f5908cc36cb22eb3c6c04f183994104
420bf1a29bc9acf8f8d86c5bb00edf70aa98edaf
6162 F20110218_AACNPJ franco_c_Page_077thm.jpg
57546a2f3fc0bed337b70f85d6c819e4
5673c0ff3fd72d365777504cd3327a4c4baa8810
6071 F20110218_AACNOU franco_c_Page_059thm.jpg
20031fb7ad4d50a85e558e5281ce6941
3d61af369a983ab064e9d022d5db07258be0b6c9
F20110218_AACMLS franco_c_Page_022.tif
bffec6da3cc49640d54057068a85a004
6b0e392663596657f8d1225ad173614b7cfe73e3
6508 F20110218_AACNPK franco_c_Page_078thm.jpg
32239cd1dd9ee9f580ecf378fc1f2f49
fb74cc4fcf4251abbc977555a9b2298e6066b0c0
6314 F20110218_AACNOV franco_c_Page_060thm.jpg
b374acacc819519a4afdc44bac42e50c
0536ec4c96d6ece300880a98c9637e6f6aa0aeed
F20110218_AACMMI franco_c_Page_001.tif
660b0ccad4a5885840861636effc956f
d8d9cc0a7d72612b75f53e589d82db4ea7aac349
21352 F20110218_AACMLT franco_c_Page_062.QC.jpg
b492c31c54bdde025f0e9e988991b868
8235ff2ea17543025e3aa8ee36c300973dd13fba
1101 F20110218_AACNPL franco_c_Page_079thm.jpg
96ee5a5722783b08514f5d554b902597
a97beb7876047bbfcfea733da48ae98b506fc8da
4636 F20110218_AACNOW franco_c_Page_061thm.jpg
7eb9d836212bc182919675e3a9c4d809
637de7eab6b8b849bc21ca5c2e97c76c355bc348
F20110218_AACMMJ franco_c_Page_003.tif
3abb4852377aa2b37391fc5aeb2feaaa
d0ba44fe1fd44b12fb26bf4ea3073e75f5c1073b
6498 F20110218_AACMLU franco_c_Page_110thm.jpg
7df4fb0cc6c806dedb467de31d2a4a61
1a5e884560bd47b3d9c7f7c44472f7b64470aa01



PAGE 1

STRATEGIES TO ENHANCE FERTILIT Y IN DAIRY CATTLE DURING SUMMER INCLUDING USE OF CRYOPRESERVAT ION OF IN VITRO PRODUCED EMBRYOS By C. MOIS S FRANCO A THESIS PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLOR IDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE UNIVERSITY OF FLORIDA 2006

PAGE 2

Copyright 2006 by C. Moiss Franco

PAGE 3

This professional achievement reflects the sacr ifice and guidance of my family especially that of my mother, Mercedes Y. Vaca El-H age, who laid the foundations with strong pillars in my life. This dissertation is dedicated to my be loved son Talyn Izaak Franco Benton and astonishing father Antonio Vicente Franco M onasterio (†) for their endless love, support and most important, inspiration. “EL HOMBRE SE AUTORREALIZA EN LA MISMA MEDIDA EN QUE SE COMPROMETE AL CUMPLIMIENTO DEL SENTIDO DE SU VIDA” Victor Frankl (1905-1997)

PAGE 4

iv ACKNOWLEDGMENTS This thesis would not have been po ssible without the enthusiasm, knowledge, guidance, tenacity, and, perhaps most importantly faith that I received from my academic advisor, Peter J. Hansen. From the very fi rst interview to the last queries on research accomplishments and career plans, he was always eager to entertain my ideas in hope that I fulfilled my dream(s) and become successful. I was not sure I could handle an undertaking of such a magnitude, but was able to thanks to hi s consistent effort and true desire to keep me on track. I would like to extend my sincere appreci ation to my committee member Dr. Karen Moore, for her insight and willingness to help me academically without fail and regards to time. Despite having other major responsibil ities, Dr. Carlos Risco was willing to help whenever asked. I thank him for his assistance and especially for the desire to help me learn to palpate. Thanks are also extende d to Dr. Alvin Warnick for his advice and suggestions for improving my research projec ts and academic training. I would also like to thank Dr. Joel Yelich for his teaching, support, and enthusiasm while providing me with ideas that can help me achieve my goals. Special thanks are extended to my family for encouraging me to seek for myself a demanding and meaningful education. This thes is could not have taken place without that precious gift. Most sincere appreciation is also due to to my colleague and friend Dr. Roco M. Rivera, whose willingness to assist me in my ea rly stages as a master's student helped to

PAGE 5

v kindle my interest in this expl oration. I would not have gotte n this far if it was not for her unique and excellent training doing IVF. Dr. Zv i Roth was an inspirational friend whose passion for science was transmitted to me. He also expressed his kindness and love towards my son. I also thank Dr. Joel Hernandez for his support, friendship, and guidance. Thanks are given to Maria B. Padua for he r assistance with the completion of this manuscript and Luis Augusto Castro e Paula. Their unconditional fr iendship and help at any given time is sincerely appreciated. I am grateful to Dean Jousan for making the time to proofread my writings throughout th e years and for his assistance in various research experiments. Special thanks go to Amber Brad for her personality and joy that helped the lab be united. Best of all has b een my colleague and friend Jeremy Block for his patience, expertise and engaging convers ations that helped develop in me new dreams. In addition, he always remained motiv ated throughout my transfer experiments. I also would like to thank Central Pa cking Co. management and personnel at Center Hill, FL, for providing the ovaries used for various experiments and William Rembert for his assistance in collecting these ovaries. Special thanks go to Mary Russell and Elise Griffin, for their assistance at the Un iversity of Florida Dairy Research Unit. I thank Luther White and Mark Saulter of H illtop Dairy, R.D. Skelton and Mathew Steed of Levy County Dairy, and Mauricio Franco and Faby Grisel of Sausalito Dairy for cooperation and assistance with th e projects. And last but not least, I would like to thank Todd Bilby, Osiloam Gomez, Reinaldo Cooke, Patrick Thompson, Saban Tekin, and Paolette Soto.

PAGE 6

vi TABLE OF CONTENTS page ACKNOWLEDGMENTS.................................................................................................iv LIST OF TABLES.............................................................................................................ix LIST OF FIGURES.............................................................................................................x ABSTRACT....................................................................................................................... xi CHAPTER 1 REVIEW OF LITERATURE.......................................................................................1 Infertility in Modern Dairy Cattle.................................................................................1 Causes for the Decline in Fertility in Dairy Cattle.......................................................2 Milk Yield.............................................................................................................2 Milk yield and energy balance.......................................................................3 Milk yield and endocrine milieu....................................................................5 Milk yield and heat stress...............................................................................6 Milk yield and diseases..................................................................................9 Milk yield, estrus detection, and fertility.....................................................10 Changes in Herd Size as a F actor in Reduced Fertility.......................................11 Inbreeding............................................................................................................12 Strategies to Improve Fertil ity in Lactating Dairy Cattle...........................................12 Treatment with Bovine Somatotropin (bST) to Enhance Fertility......................13 Treatment with GnRH to Delay Luteolysis.........................................................14 Increase in the Size of the Preovulatory Follicle to Generate a Larger Corpus Luteum.............................................................................................................17 Induction of an Accessory Corpus Luteum.........................................................19 Progesterone Supplementation............................................................................20 Inhibition of Luteolysis.......................................................................................21 Nutritional Strategies...........................................................................................22 Fat feeding to improve energy balance........................................................22 Administration of antioxidants.....................................................................25 Crossbreeding......................................................................................................26 Embryo Transfer..................................................................................................27 Limitations to Optimal Pregnancy Rates Using IVP TET................................28 Cryopreservation of IVP Embryos......................................................................30 Summary and Objectives of the Thesis......................................................................31

PAGE 7

vii 2 EFFECTIVENESS OF ADMINIS TRATION OF GONADOTROPIN RELEASING HORMONE AT DAY 11, 14 OR 15 AFTER ANTICIPATED OVULATION FOR INCREASING FERTILITY OF LACTATING DAIRY COWS AND NON-LACTATING HEIFERS............................................................34 Introduction.................................................................................................................34 Materials and Methods...............................................................................................35 Experiment 1 GnRH Administration at Day 11 after Anticipated Ovulation in Heifers Subjected to Timed Artific ial Insemination during Heat Stress.....35 Experiment 2 GnRH Administration at Day 11 after Anticipated Ovulation in Lactating Cows Subjected to Timed Artificial Insemination......................37 Experiment 3 GnRH Administration at Day 14 after Anticipated Ovulation in Lactating Cows Subjected to Timed Artificial Insemination......................38 Experiment 4 GnRH Administration at Day 14 after Anticipated Ovulation in Lactating Cows Subjected to Time d Artificial Insemination During Heat Stress................................................................................................................39 Experiment 5 GnRH Administration at Day 14 or Day 15 after Detected Estrus................................................................................................................40 Statistical Analysis..............................................................................................40 Results........................................................................................................................ .42 Experiment 1 GnRH Administration at Day 11 after Anticipated Ovulation in Heifers Subjected to Timed Artific ial Insemination During Heat Stress....42 Experiment 2 GnRH administration at Day 11 after Anticipated Ovulation in Lactating Cows Subjected to Timed Artificial Insemination......................42 Experiment 3 GnRH Administration at Day 14 after Anticipated Ovulation in Lactating Cows Subjected to Timed Artificial Insemination......................43 Experiment 4 GnRH Administration at Day 14 after Anticipated Ovulation in Lactating Cows Subjected to Time d Artificial Insemination During Heat Stress................................................................................................................43 Experiment 5 GnRH Administration at Day 14 or Day 15 after Detected Estrus................................................................................................................44 Overall Effectiveness of GnRH Treatme nt as Determined by Meta-Analysis....44 Discussion...................................................................................................................44 3 EFFECT OF TRANSFER OF ONE OR TWO IN VITRO-PRODUCED EMBRYOS AND POST-TRANSFER ADMINISTRATION OF GONADOTROPIN RELEASING HORMONE ON PREGNANCY RATES OF HEAT-STRESSED DAIRY CATTLE.......................................................................52 Introduction.................................................................................................................52 Materials and Methods...............................................................................................54 Experiment 1 Single or Twin Transf er of IVP Embryos into Crossbred Dairy Recipients...............................................................................................54 Experiment 2 Administration of GnRH on Day 11 after Anticipated Ovulation in Lactating Recipients that Received an IVP Embryo..................57 Statistical Analysis..............................................................................................59 Results........................................................................................................................ .60

PAGE 8

viii Experiment 1 Single or twin transfer of IVP embryos......................................60 Pregnancy and calving rates.........................................................................60 Characteristics of gestati on, parturition, and calves.....................................61 Experiment 2 Administration of GnRH on Day 11 after Anticipated Ovulation..........................................................................................................62 Discussion...................................................................................................................62 4 EFFECTS OF HYALURONIC ACID IN CULTURE AND CYTOCHALASIN B TREATMENT BEFORE FREEZING ON SURVIVAL OF CRYOPRESERVED BOVINE EMBRYOS PROD UCED IN VITRO........................................................72 Introduction.................................................................................................................72 Materials and Methods...............................................................................................73 Embryo Production..............................................................................................73 Experimental Design and Embryo Manipulation................................................74 Cryopreservation.................................................................................................75 Thawing and Determination of Survival.............................................................76 Statistical Analysis..............................................................................................76 Results........................................................................................................................ .77 Effect of Hyaluronic Acid on Embryonic Development.....................................77 Survival after Cryopreservation..........................................................................77 Discussion...................................................................................................................78 5 GENERAL DISCUSSION.........................................................................................82 LIST OF REFERENCES...................................................................................................91 BIOGRAPHICAL SKETCH...........................................................................................123

PAGE 9

ix LIST OF TABLES Table page 2-1 Descriptive statistics, adjusted odds ratio (AOR) estimates, and 95% Wald confidence intervals (CI) for effect of GnRH administrati on at Day 11 after anticipated ovulation and ovul ation synchronization prot ocol on pregnancy rates of heifers during heat stress......................................................................................49 2-2 Descriptive statistics, adjusted odds ratio (AOR) estimates, and 95% Wald confidence intervals (CI) for effect of GnRH administrati on at Day 11 after anticipated ovulation and s eason of insemination on pregnancy rates of lactating cows subjected to timed artificial insemination.......................................................50 2-3 Descriptive statistics, adjusted odds ratio (AOR) estimates, and 95% Wald confidence intervals (CI) for effect of GnRH administrati on at Day 14 after anticipated ovulation and s eason of insemination on pregnancy rates of lactating cows subjected to timed artificial insemination.......................................................50 2-4 Descriptive statistics, adjusted odds ratio (AOR) estimates, and 95% Wald confidence intervals (CI) for effect of GnRH administrati on at Day 14 after anticipated ovulation and Da ys in milk (<150 d vs > 150) at insemination on pregnancy rates of lactating cows subj ected to timed artificial insemination during heat stress......................................................................................................51 3-1 Effect of recipient type and number of embryos transferred per recipient on pregnancy rates and losses.......................................................................................68 3-2 Effect of recipient type and number of embryos transferred per recipient on characteristics of pregnancy and parturition............................................................69 3-3 Effect of recipient type and number of embryos transferred per recipient on characteristics of calves born...................................................................................70 4-1 Effect of hyaluronic acid added at day 5 after insemination on production of blastocysts at day 7 a nd 8 after insemination. .........................................................81 4-2 Effect of culture in hyaluronic acid and treatment with cytochalasin B on survival after cryopreservation. ...............................................................................81

PAGE 10

x LIST OF FIGURES Figure page 1-1 Rolling herd average (RHA, kg milk pe r lactation), calving interval (CI), and services per conception (SPC) for 143 da iry herds continuously enrolled in the Raleigh DHIA record system from 1970 to 1999. ..................................................32 1-2 Temporal changes in first service pregnancy rate and annual average milk production from high-producing Holstein-Fri esian dairy herds in north-eastern Spain. Data for pregnancy rate were r ecorded in the cool (October April months) and warm season (May-September months). ...........................................33 3-1 Maximum (open circles) and minimum (c losed circles) daily air temperatures and relative humidities (RH) during the experiments..............................................71

PAGE 11

xi Abstract of Thesis Presen ted to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Master of Science STRATEGIES TO ENHANCE FERTILIT Y IN DAIRY CATTLE DURING SUMMER INCLUDING USE OF CRYOPRESERVAT ION OF IN VITRO PRODUCED EMBRYOS By C. Moiss Franco Vaca May 2006 Chair: Peter J. Hansen Major Department: Animal Sciences There has been a precipitous decline in fer tility of lactating dair y cows. In addition, heat stress can further compromise fertility. The goals of this thesis were to 1) evaluate strategies for enhancing fer tility after artificial insemi nation using mid-cycle GnRH treatment and 2) further develop embryo tran sfer using in vitro produced embryos as a tool for increasing fertility. For the sec ond objective, experime nts tested whether pregnancy rate could be improved by tran sfer of twin embryos and whether the developmental competence of embryos afte r cryopreservation could be improved by hyaluronic acid or cytochalasin B treatment. A series of six experiments were conduc ted to test the efficacy of GnRH for increasing fertility. Except for one experime nt, in which GnRH administration at day 14 after insemination increased pregnancy rate, GnRH was without effect whether given at

PAGE 12

xii day 11, 14 or 15 after insemination or at da y 11 after anticipated ovulation in embryo transfer recipients.. Neither unilateral transfer of two embr yos nor administration of GnRH at Day 11 after anticipated ovulation improved pregnanc y rates of dairy cattle exposed to heat stress. Cytochalasin B treatment before freezing improved cryosurvival of bovine embryos produced in vitro. In contrast, cu lture with hyaluronic acid was of minimal benefit. Taken together, GnRH treatment did not consistently increa se pregnancy rates when administered at Day 11-15 after insemina tion and is not recommended as a fertilityenhancing treatment. Similarly, tr ansfer of two embryos to th e uterine horn ipsilateral to the CL was not an effective method for increas ing pregnancy rates in recipients. Transfer of cryopreserved embryos may be enhanced by treatment of embryos with cytochalasin B since this molecule increased in vitro surv ival, and it remains to be tested whether survival of IVP embryos afte r vitrification can be improve d by cytochalasin B treatment.

PAGE 13

1 CHAPTER 1 REVIEW OF LITERATURE Infertility in Modern Dairy Cattle Fertility is defined as the ability of a cycl ic animal to establish pregnancy and is an important economic trait that a ffects herd productivity in da iry cattle (Pecsok et al., 1994; Plaizier et al., 1998). Unfortuna tely, there has been a decline in fertility in dairy cows over the last 10-40 years. Fertility, whethe r traditionally measured as conception rate (number of pregnant animals divided by the number of inseminated animals) or herd pregnancy rate (number of pregnant animals di vided by the number of animals eligible to be bred), has declined in North Ameri ca (Butler, 1998), Ireland (Roche, 2000), Spain (Lpez-Gatius, 2003), and the United Kingdom (Royal et al., 2000). Other important reproductive measurements have changed during this time as well, including increases in days to first service, days to conception, and calving interv al (de Vries and Risco, 2005). The magnitude of these changes in reproducti ve function over time is illustrated for data from herds in the United States (Figure 11) and northeastern Sp ain (Figure 1-2). The incidence of infertility of dairy cows has been correlated with changes in dairy cattle physiology and improvements in gene tic progress, nutrition, and management practices. This litera ture review will seek to identi fy physiological causes for this decrease in fertility and describe efforts to improve fertility.

PAGE 14

2 Causes for the Decline in Fertility in Dairy Cattle Milk Yield The Animal Improvement Programs Laborator y of the United States Department of Agriculture (USDA) has estimated the genetic trend for milk yield with an average of 37 kg/yr during the 1960s, 79 kg/yr during th e 1970s, 102 kg/yr during the 1980s, and 116 kg/yr for the period from 1990 to 1996 ( http://aipl.arsusda.gov ; Hansen, 2000). It has long been known that fertility is reduced in lactating cows as compared to non-lactating heifers (Ron et al., 1984; Ne bel and McGilliard, 1993). Gi ven that milk yield has increased over time as fertility has declined, the possibility must be considered that the increase in milk yield is one reason that has contributed to the decreased fertility in dairy cattle. There are indications that the genetic correlation between female fertility and milk production is antagonistic (Kadarmideen et al ., 2000; Royal et al., 2002). In contrast, Mahanna et al. (1979) suggested that there was no negative genetic correl ation between milk yield and reproduction because there wa s no difference in fertility among heifers with different genetic abilities for milk yield. There may be an environmental effect of milk yield on fertility, however. As describe d by Lucy (2001), the increase in milk yield over the period from 1970 has been associated with a corresponding decrease in fertility as measured by increased services per con ception and calving interval (Figure 1-1). According to Nebel and McGilliard (1993) ther e was little or no asso ciation of increased milk yield compromising fertility prior to the 1970s (Gaines, 1927; Boyd et al., 1954; Currie, 1956; Smith and Legates, 1962) but a dverse effects of milk yield have been correlated with reduced fertility in studie s conducted since 1975 (S palding et al., 1975;

PAGE 15

3 Laben et al., 1982; Fonseca et al., 1983; St evenson et al., 1983; Hillers et al., 1984; Wiggans et al., 1987; Faust et al., 1988). Using a data set of Holstein, Jersey, and Guernsey cows, it was found that 0.014 more services per conception were required for each additional 100 kg of 120-d milk for Holsteins and 0.028 services per conception for Jersey and Guernsey cows (Olds et al., 1979). Similarly, cows with the highest milk yield had the lowest first service conception rate (Faust et al., 1988) or 90-d non-return rate (Al-Katana ni et al., 1999) and highest number of services (Faust et al., 1988). Days to first in semination and days open also increased linearly as milk yield increased in Jersey dairy cattle (Fonseca et al., 1983). Expression of estrus at first postpartum ovulation is less li kely in cows with higher milk production (Westwood et al., 2002). Some st udies (Nielen et al., 1989; Kinsel et al., 1998), but not others (Deluyker et al., 1991), co rrelate the incidence of twins to milk yield. Amount of milk yiel d, however, was not correlated to increased incidence of multiple ovulations (Lpez-Gatius et al., 2005b), yet the incidence of double ovulations and twinning rate has increased in modern dairy cattle (Wiltbank et al., 2000). Taken together, the associations of milk yield with reduced duration of estr us, increased days to first insemination, increased number of in seminations per conception, reduced first service conception rate s, and reduced progesterone le vels post-ovulation compromise herd fertility. Milk yield and energy balance One way in which milk yield could aff ect fertility is through effects on energy balance. A critical phase exists in the period following calving when dry matter intake does not meet the increased metabolic demands of lactation, and as a result, the animal

PAGE 16

4 enters a state classified as “negative ener gy balance” (NEB). Duri ng the period of NEB, body reserves of fat and protein are mobili zed (Bauman and Currie, 1980; Butler and Smith, 1989). An animal under NEB tends to have low body condition score (BCS), and both NEB and low BCS are associated with low fertility (O’Ca llaghan, 1999; Butler, 2000; Pryce et al., 2001; Pus hpakumara et al., 2003). Energy deficiency reduces or impairs gona dotropin secretion, a nd as an animal reaches this state around parturition, gonadot ropin secretion to support follicular development and ovulation is compromise d and reproductive problems (i.e., cystic ovaries) associated with onset of ovarian activ ity become prevalent (Zulu et al., 2002ab). Growth hormone stimulates insulin-like grow th factor 1 (IGF-1) production by the liver (Jones and Clemmons, 1995), but during NEB growth hormone receptors are downregulated in a process referred to as “Growth Hormone Resistance” (Donaghy and Baxter, 1996). As milk production increases during early lactation and the cow is under NEB, the liver becomes refractory to growth hormone because growth hormone receptors are decreased (Vicini et al., 1991), and this result in reduced plasma concentration of IGF-1 (Pell et al., 1993). Follicular growth is stimulated by IGF1 (Webb et al., 2004) and reduced plasma concentrations of this growth factor are observed in cows wi th high milk yield (Rose et al., 2004) and together are highly correlated to delayed return to ovarian cyclicity (Taylor et al., 2004). After calving, cows with IGF-I concentrations gr eater than 50 ng/ml at first service were 5 times more likely to concei ve than those with lower concentrations (Taylor et al., 2004).

PAGE 17

5 The fact that high-producing cows have greater energetic demands for lactation does not necessarily mean that these cows ha ve greater NEB or low BCS. Staples et al. (1990) found that low-producing cows had lower dry matter intake and were at a greater risk for failure to conceive due to anestrus and infertility than high-producing cows. It was observed that the low-producing group, cla ssified as non-responders, sustained milk production from 28% of body tissue reserve vs 15.9 and 16.7% in the early responder and late responder groups. This interaction was confirmed when low-producing cows had lost the most body weight during the first 2 w eeks of lactation and we re in the greatest energy deficit (Staples et al., 1990). Milk yield and endocrine milieu Cows displaying greater milk producti on often have higher dry matter intakes (Staples et al., 1990; Hommeida et al., 2004), which has been demonstrated to decrease circulating progesterone concentrations in lactating (Hommeida et al., 2004) and nonlactating cows (Rabiee et al ., 2001). Acute feeding reduced circulating progesterone by 25% in pregnant cows (Vasconcelos et al., 2003). Lucy and co-workers (1998) found that circulating progesterone was lower in cattle genetically se lected for high milk production. Sangsritavong et al. (2002) demonstrated that lactating cows have a much greater steroid metabolism than non-lactating cows. As a result, lactating cows may have larger luteal tissue volume on the ovary (Sartori et al., 2002; Sartori et al., 2004) yet experience lower circulating progesterone and estradiol co ncentrations than heifers and dry cows (De la Sota et al., 1993; Wolfenson et al., 2004). There is evidence that low progesterone

PAGE 18

6 secretion can compromise fertility in da iry cattle (Mann and Lamming, 1999) and an increase in progesterone secretion ma y facilitate embryonic development. Progesterone provides nourishment for the co nceptus via induction of secretion of proteins and other molecules from the e ndometrium (Garrett et al., 1988a). Low peripheral concentrations of pr ogesterone are also associated with increased luteinizing hormone (LH) pulses (Ireland and Roche, 1982) that can stimulate lu teolytic signals in favor of pregnancy failure. Skarzynski and Okuda (1999) reporte d that blocking the progesterone receptor with a progesterone antagonist (onapristone) increased prostaglandin F2 (PGF2 ) production by bovine luteal cells harvested from mid-cycle corpora lutea (CL) (Days 8–12). In addition, it was revealed that the bovine corpus luteum (CL) does not undergo apoptosis until progesterone production has declined (Juengel et al., 1993; Rueda et al., 1995). Milk yield and heat stress One reason why milk yield might decrease fe rtility of lactating cows is because it increases their susceptibility to heat stress. Infertility is a particul ar problem during heat stress (Ingraham et al., 1974; Putney et al., 1989b; Al-Katanani et al., 1999) and air temperatures as low as 27oC can induce hyperthemia in lact ating dairy cows (Berman et al., 1985). Cows exposed to elevated temper atures to induce heat stress experienced reduced pregnancy rates (Dunlap and Vincen t, 1971) and increased embryonic mortality (Putney et al., 1988ab; Ealy et al., 1993). On the other hand, provisi on of cooling in the summer increased pregnancy rates as compared to non-cooled cows (Stott et al., 1972; Roman-Ponce et al., 1981; Ealy et al., 1994).

PAGE 19

7 The ability to regulate body temperatur e during heat stress is exacerbated by lactation because of the excess heat produc tion. The increase in body temperature in response to heat stress is great er for lactating cows than heifers (Cole and Hansen, 1993) and greater for high-producing cows than low-producing cows (Berman et al., 1985). Data collected on fertility at first service from 8124 Holstein cows located in South Georgia as well as North and South Florida support the idea that a high level of milk production reduces fertility of lactating cows When cows were grouped according to mature equivalent milk yield, there was a milk yield class x month of breeding interaction that resulted from the fact that the duration and magnitude of summer infertility increased as milk yield increased (Al-Katanani et al., 1999). Heat stress before, shortly after, and on the day of breeding is associated with reduced fertility. Heat stress can compromi se fertility throughout various reproductive processes such as oocyte developmental comp etence (Picton et al., 1998; McNatty et al., 1999) since the oocyte becomes sensitive to da mage throughout the various stages of follicular growth (Badinga et al., 1993). Indeed, follicular steroidogenesis, follicular dynamics and altered concentra tions of FSH and inhibin beco me altered in response to heat stress (Badinga et al., 1994; Wolfenson et al., 1997; Roth et al., 2000). During heat stress sperm can be damaged after insemina tion due to the generation of reactive oxygen species (Ishii et al., 2005) and embryonic de velopment can be co mpromised directly (Monty et al., 1987). Not surprisingly the heat stress problem is mu ltifactorial (Hansen et al., 2001). Heat stress of superovulated cows at day 1 after breedi ng reduced the proportion of embryos that were blastocysts at day 8 afte r breeding, but heat stress on day 3, 5 or 7

PAGE 20

8 after breeding did not affect subsequent embryonic development (Ealy et al., 1993). Superovulated heifers experien ced a high percentage of reta rded embryos recovered on day 7 after insemination after exposure to hi gh temperature and humidity at the onset of estrus for 10 h (Putney et al., 1989a). In another study heat stress was induced in Holstein heifers by submitting them from day 1 to day 7 after estrus to 42oC for 7 h (treatment) or 30oC for 16 h (control) and results obtained revealed more retarded embryos with degenerate blastomeres on the day of recovery (20.7% vs. 51.5%, respectively; Putney et al., 1988a). One cause for the observed reduction in re productive performance under heat stress conditions is steroidogenic capacity and its e ffects on oocyte function (Roth et al., 2001; Al-Katanani et al, 2002b; Roth and Hansen, 2004). Under heat st ress, low estradiol concentration in the follicular fluid of do minant follicles involves reduced aromatase activity in the granulosa cells (Badinga et al., 1993) and reduced androstenedione production by theca cells (Wolfenson et al ., 1997). Although earlier studies were inconsistent in demonstrating that plasma c oncentrations of estrad iol are reduced under heat stress (no change– Gwazdauskas et al., 1981; increase – Rosenberg et al., 1982; decrease – Gwazdauskas et al., 1981), recent work points toward heat stress resulting in lower estradiol concentrations in the follicular fluid (Badinga et al., 1993; Wolfenson et al., 1995; Roth, 1998; Wilson et al., 1998ab). Heat stress also has been reported to d ecrease (Rosenberg et al., 1982, Younas et al., 1993; Howell et al., 1994), in crease (Abilay et al., 1975; Roman-Ponce et al., 1981; Trout et al., 1998), or have no effect (Wise et al., 1988; Wolfenson et al., 1995) on peripheral concentrations of progesterone. Elev ated temperatures in culture can directly

PAGE 21

9 influence endometrium explants by increasing PGF2 secretion (Putney et al., 1988c; Malayer and Hansen, 1990) and from days 816 of pregnancy can reduce the size of the embryo at day 17 (Biggers et al., 1987). A retrospective survey involving 12,711 lact ations from high-yielding dairy herds in northeast Spain demonstrat ed that milk yield per cow increased from 1991-2000 (Lpez-Gatius, 2003; see Figure 2). For each 1000 kg increase in average milk yield in the warm period, there was a decrease of 6% in pregnancy rate, and 7.6% in cyclicity, and an increase of 8% in the incidence of inactive ovaries. During the cool period, however, there was no change in fertility over time. Thus, the continual increase in milk yield might have reduced fertility in Spain, at least, by exacerba ting effects of heat stress. Milk yield and diseases Increased incidence of certain diseases has been associated with elevated milk yield. High somatic cell score and clinical mastitis (Schukken et al., 1990; Barkema et al., 1998; Chassagne et al., 1998; Fleischer et al., 2001); lameness (Green et al., 2002); cystic ovarian disease (Fleis cher et al., 2001; Lpez-Ga tius et al., 2002); milk fever (Fleischer et al., 2001); and acu te metritis (Kelton et al., 1998) are all co rrelated with milk yield. Compared to non-mastitic herd-mates, high producing cows were at a greater risk of developing clinical mastitis (Gr hn et al., 2004). Number of days to conception, artificial inseminations per conception and num ber of days to first artificial insemination (AI) were significantly greater for cows with clinical mastitis (B arker et al., 1998), and may affect embryonic survival when occurr ing after insemination (Soto et al., 2003). According to Jousan et al., (2005) an elev ated somatic cell count score among lactating

PAGE 22

10 females influenced mid-to-late fetal loss (re presented as occurring after day 70 to 90 of gestation) and mastitis has been reported to affect pregnancy lo ss during the period of embryonic (Chebel et al., 2004) and fetal develo pment (Risco et al., 1999; Santos et al., 2004a). High yielding cows had an increased likelihood of becoming lame (Green et al., 2002) and cows that had been treated for la meness had a negative influence on pregnancy to first insemination and numbers of insemi nations per service period (Petersson et al., 2005). Similarly, non-lame cows were more likel y to conceive at first service than lame cows and lameness within the first 30 days after calving was associated with reduced pregnancy rates at first AI a nd a higher number of services per conception (Hernandez et al., 2001; Melendez et al., 2003). In a meta-a nalysis of several published papers, leg problems were associated with an average increase of 12 days to conception (Fourichon et al., 2000). Cows that develop cysts remain infertile as long as this conditi on persists and early spontaneous cyst recovery was negatively corr elated with milk yiel d (Lpez-Gatius et al., 2002). Similarly, elevated milk yield increase d the risk of cows developing cysts (LpezGatius et al., 2002) and days from metritis o ccurrence to first AI is also correlated to infertility (Loeffler et al., 1999) Milk yield in the current lact ation is also correlated with incidence of milk fever (Fleis cher et al., 2001) and this dis ease reduces fertility (Chebel et al., 2004). Milk yield, estrus de tection, and fertility Milk yield may affect fertility indirectly by reducing the ability to accurately detect estrus. An antagonistic rela tionship between increased milk production and days to first

PAGE 23

11 visual estrus has already b een reported. According to Lpez et al. (2004), duration, standing events, intensity (determined by th e number of standing events per hour), and standing time were reduced for high-producing cows as compared to low producers. Similarly, Harrison et al. (1990) reported that elevated milk yield was correlated to a longer period of estrus suppr ession. Westwood et al., (2002) indicated that high genetic merit for milk yield influenced significan tly the chance a cow showed weak signs of estrus as compared to lo w milk producing cows. Cows with elevated milk yield also had re duced circulating estr adiol concentrations on the day of estrus expression and shorter duration of estrus de spite having larger preovulatory follicle diameters (Lpez et al., 2004). Changes in Herd Size as a Factor in Reduced Fertility Increased milk yield is not the only change in dairy farming over the last 50 years and some of these other changes could also contribute to decreased fertility. One major change has been the trend towards large farms. In a review, Lucy et al. (2001) cited data from the USDA National Agricultural Statistics Services that nearly 30% of all dairy farms in the United States have more than 500 cows. In additi on, Stahl et al. (1999) reported that the expansion of dairy herds comes in large part through the purchase of first-lactation cows. Thus, as Lucy et al (2001) pointed out, these more infertile primiparous cows (Stahl et al., 1999) may ha ve represented an increasingly larger percentage of the herd as dairy herds have expanded ove r the last 10-40 years. The importance of changes in herd size as a cause for infertility have been questioned by de Vries and Risco (2005) who found no clear association with reproductive function. Nevertheless, as the herd size is increased one would expect th at the likelihood that it becomes harder for accurately detecting estrus becomes a challenge because factors

PAGE 24

12 associated with herd size such as the su rface (concrete floor) on which the cow stands will reduce the preponderance of cows displa ying estrus activity (Britt et al., 1986; O’Connor and Senger, 1997). Inbreeding Inbreeding represents increased frequency of identical al leles at a gene locus and the inbreeding percent is a measure for the ge nes of an individual that are identical by descent (Wright, 1922; Falconer, 1981). It is generally considered that reproductive function declines when inbreed ing levels in a population ri se above 6.25% (Hansen et al., 2005). Increased degree of inbreeding as the result of use of AI could explain some of the declines in fertility experienced by dairy cattle because inbreeding coefficients have increased in all the major U.S. dairy breeds. Estimates of inbreedi ng in the U.S. dairy population are near 5% currently (Short et al., 1992; Wiggans et al., 1995; Young et al., 1996; Hansen, 2000; Wall et al., 2005) and increa sing at a constant ra te of about 0.1% per year for U.S. Holsteins (Hansen et al., 2005). At an average of 5%, it is likely that many dairy cows have inbreeding coefficients above 6.25% (Hansen et al., 2005). Thompson et al. (2000ab) found calving inte rvals to increase by 12 and 17 d for Jersey and Holsteins cows, respectively, with levels of inbreeding >10%. Similarly, inbreeding had pronounced negati ve effects on fertility at higher levels (10%) of inbreeding (Wall et al., 2005). In another study, animals with an inbreeding coefficient >9% had fewer transferable embryos following superovulation than animals with a lower inbreeding coefficient (Alvarez et al., 2005). Strategies to Improve Ferti lity in Lactating Dairy Cattle Four general approaches to improve repr oductive function in dairy cattle have been developed. The first is to regula te the timing of ovulation using gonadotropin

PAGE 25

13 releasing hormone (GnRH) and PGF2 utiliz ed in timed AI (TAI) programs. The advantage of this approach is that this program maximizes the number of animals inseminated and allows inseminations to be made at some pre-planned time to eliminate the need for estrus detection. Pioneering st udies (Thatcher et al ., 1989; Twagiramungu et al., 1992; Wolfenson et al., 1994) were able to synchronize estrus effectively, however, subsequent studies at the University of Fl orida (Schmitt et al., 1996a) and University of Wisconsin (Pursley et al., 1995) led to the development of the Ov synch TAI program and the demonstration that good pregnancy rate s can be achieved (Thatcher et al., 2001; Thatcher et al., 2002). Although th is approach is an effective one and is widely used in dairy herds, it involves regul ation of events occurring be fore conception and is beyond the scope of the present review The second approach is to use information regarding the hormonal basis for establishment of pregna ncy and signaling between the maternal and embryonic units during early pregnancy as th e basis for pharmacological treatments to improve embryonic survival. Failure of essential bioche mical dialogue between the conceptus and the maternal unit undoubtedly contributes to embryonic mortality and termination of pregnancy (Spencer et al ., 1996; Spencer and Bazer, 2002). The third approach has been to regulate the nutrition of the dairy cow to improve energy balance or to provide specific nutrients that favor esta blishment and maintena nce of pregnancy. Finally, recent work has focused on use of embryo transfer to bypass early embryonic death and perhaps coupled with crossbreeding may become an important alternative since Holsteins have become more inbred (Hansen et al., 2005). Treatment with Bovine Somatotrop in (bST) to Enhance Fertility Circulating concentrations of IGF-I, glucose, and c holesterol are reduced in lactating animals (de la Sota et al., 1 993; Beam and Butler 1997). Circulating

PAGE 26

14 concentrations of IGF-I is influenced by nut rition (Adam et al., 1997) and closely related to energy balance of the cow (Ginger et al ., 1997; Beam and Butler, 1998; 1999). Present in serum and in various tissues, IGF-I is produ ced mainly by the liver but other organs as well (Murphy et al., 1987; Thi ssen et al., 1994). IGF-I regula tes ovarian function in dairy cattle (Breukink et al., 1998; Chase et al ., 1998), is necessary for proper follicular development in which a fully competent ooc yte capable of inducing ovulation develops (Lucy et al., 1992a), and is required for nor mal CL formation and function (Leeuwenberg et al., 1996; Chase et al., 1998). Dairy cows that initiated estrous cyclicity during the postpartum period had higher plasma IGF-I than anestrous cows (Thatcher et al., 1996), cystic and inactive ovary or persistent CL cows (Zulu et al., 2002a). Bovine somatotropin (bST) increases plasma concentrations of insulin, IGF-I, and growth hormone (Bilby et al., 2004), perhaps by stimulating ovarian function especially after IGF-1 plasma levels are reduced in lact ating animals (de la Sota et al., 1993). In addition, injection of bST stimulates concep tus growth by day 17 of pregnancy (Bilby et al., 2004). Additional studies provided evidence that bST can improve pregnancy rates in lactating cows (Moreira et al ., 2000b; Morales-Roura et al., 2001; Santos et al., 2004b). Superovulated donor cows that received bST treatment experienced reduced number of unfertilized oocytes, increased number of embryos that developed to the blastocyst stage, and increased number of transferable embryos (Mor eira et al., 2002). Collectively, these studies indicate that critical thresholds of GH and IGF-I concentr ations are needed to stimulate reproductive performance (Bilby et al., 2004). Treatment with GnRH to Delay Luteolysis The estrous cycle is characterized by 2, 3, and sometimes 4 waves of follicular growth (Sirois and Fortune, 1988; Ginther et al., 1996). During the second half of the

PAGE 27

15 luteal phase, development of an estrogenic foll icle facilitates the luteolytic process via secretion of estradiol. Nonpregnant cows have higher pe ripheral concentrations of estradiol on days 16 and 18 after breeding comp ared to pregnant animals (Ahmad et al., 1997). Thatcher et al. (1991) examined the la rgest and second larges t follicles present on day 17 after estrus in pregnant and cyclic dairy cows. In the cyclic cows, the largest follicle had greater aromatase activity and c ontained more estradiol and less progesterone in the follicular fluid than the second larges t follicle. These relati onships were reversed in pregnant animals, which indicated an earlier recruitment of the third wave of follicular development in the pregnant animal associ ated with delayed luteolysis and higher pregnancy rates. That these follicles play an important role in luteolysis was shown by Villa-Godey et al. (1985), who re ported that electrocautery to destroy large follicles was associated with an extension of the estrous cycle. Estradiol is now known to be one of three hormones that contro l uterine secretion of PGF2 with progesterone and oxytocin also bei ng involved. Pulsatile release of PGF2 from the luminal epithelium of the endometr ium is stimulated via oxytocin (Roberts and McCracken, 1976; Silvia and Taylor, 1989; Mi lvae and Hansel, 1980). Progesterone and estradiol regulate this process because estr adiol induces formation of oxytocin receptors (Silvia and Taylor, 1989; Zi ngg et al., 1995; Robinson et al ., 2001) after progesterone exposure (Ginther, 1970; Garrett et al ., 1988b; Lafrance and Goff, 1988). While progesterone initially suppresses PGF2 secretion by blocking oxytocin receptors during the early and mid-luteal phase of the estrous cycle, the e ndometrium becomes responsive to oxytocin and progesterone receptors become down regulat ed as the estrous cycle progresses (Lafrance and Goff, 198 8; Spencer and Bazer, 1995).

PAGE 28

16 Delaying luteolysis might improve pregnanc y rate by allowing embryos more time to produce sufficient quantities of interferon(IFN). Eliminating or decreasing estradiol production from the dominant follicle during the critical period of early pregnancy could be one strate gy to improve pregnancy establ ishment (Thatcher et al., 2000; Binelli et al., 2001). One approach fo r doing this is to use GnRH to regulate follicular function. Gonadotropin releasing hormone is a decap eptide that plays a central role in regulating reproductive processe s. Release of GnRH from the hypothalamus occurs in a pulsatile fashion and can be regulated by va rious internal and external signals. Hypothalamic GnRH is synthesized in cell bodies of neurosecretory neurons, and is transported to and released from th e median eminence into the hypothalamichypophyseal portal system (Loucopoulos and Feri n, 1984). GnRH has its primary effects at the pituitary gonadotrope a nd stimulates the pulsatile release of the gonadotropins luteinizing hormone (LH) and follicle-stim ulating hormone (FSH) into the peripheral circulation (Chenault et al., 1990). Two potential gonadotropi n responsive tissues within the ovary are the CL and the follicle. LH re lease induces ovulation or luteinization of large ovarian follicles present at the time of treatment (Thatcher and Chenault, 1976). One strategy tested for in creasing pregnancy rate is to inject GnRH or GnRH analogues at day 11-14 after estrus to increa se progesterone secretion (Willard et al., 2003) and delay luteolysis (Macmillan and Thatcher, 1991), thereby increasing the chance for an embryo to initiate its own antiluteolytic mechanism. Injection of GnRH at this time can lead to decreased estrogen secretion (Rettmer et al., 1992a; Mann and

PAGE 29

17 Lamming, 1995a) in an action that likely involves luteinizat ion of the dominant follicle (Thatcher et al., 1989; Rettmer et al., 1992a; Ryan et al., 1994). Improvement of fertility has been seen by administration of GnRH or its analogues at day 11-14 in nulliparous beef heifers (R ettmer et al., 1992b) and lactating dairy cows (Macmillan et al., 1986; Lajili et al., 1991; Sh eldon and Dobson, 1993; Drew and Peters, 1994; Willard et al., 2003; Lpez-Gatius et al., 2005a). In contrast to these positive results, there was no favorable effect of simila r treatments of GnRH or GnRH analogues on pregnancy rates in other studies (Jubb et al ., 1990; Stevenson et al ., 1993; Ryan et al., 1994; Bartolome et al., 2005). In a meta-analysi s of published results, Peters et al. (2000) concluded that the overall effect of GnRH administration between day 11 and 14 after anticipated ovulation was positive but that resu lts were not consistent between studies. Increase in the Size of the Preovulatory Follicle to Generate a Larger Corpus Luteum As mentioned earlier, high-yielding dair y cows are more likely to have lower circulating concentrations of progesterone th roughout the estrous cycle than cows with lower milk yields because of increased rate of progesterone catabolism (Lucy et al., 1998; Vasconcelos et al., 1999). Given the impor tance of progesterone concentration for embryonic survival (Man and Lamming, 2001), efforts have been made to increase progesterone secretion in cows. One possible e ffect of mid-cycle trea tment with GnRH is to increase progesterone secretion (Schmitt et al., 1996b; Willard et al., 2003). Another approach for increasing progester one concentrations has been to regulate the size of the preovulatory follicle to affect subsequent CL function. Optimum differentiation and growth rate of the CL varies according to the duration and amplitude of the ovulatory LH surge such that inhibition of LH release preceding the

PAGE 30

18 preovulatory surge of LH result ed in development of a smaller CL in diameter (QuintalFranco et al., 1999). Induced ovulation of sm all follicles resulted in a smaller CL and reduced secretion of progesterone than when a larger follicle ovulat ed (Vasconcelos et al., 2001). In another study (Perry et al, 2005), regression analys is indicated that pregnancy rate for cows with induced ovulat ion with an ovulating follicle of 14.5 mm was higher than for cows ovulating follicles <10.3 mm in diameter. It was further revealed that 39% of cows that lost their pregnancy had ovulatory follicles < 11 mm in diameter. Among cows that ovulated spontan eously, however, pregnancy rates at day 27 and 68 were independent of ovulatory follicle size (Perry et al., 2005). In contrast to this result, Vasconcelos et al (1999) found that the group of cows ovulating larger follicles had lower pregnancy rates on day 28 and 98 af ter AI and higher pregnancy loss between these times. Administration of GnRH just prior to or at the time of the LH surge causes an amplified preovulatory surge of LH (Lucy a nd Stevenson, 1986; Yoshioka et al., 2001). Injection of GnRH at or near the time of estrus increased the proportion of large luteal cells in the CL on day 10 of the estrous cycl e (Mee et al., 1993), pe ripheral progesterone concentrations during the first 7 days of the estrous cycle (Lucy and Stevenson, 1986), and increased pregnancy rates in repeat breed ing cows (Stevenson et al., 1990; Mee et al., 1993). Ullah et al. (1996) observed th at GnRH treatment at estrus in dairy cows improved pregnancy rates and increased peripheral progesterone con centration. Conversely, GnRH administered to lactating dairy cows at th e time of AI did not a ffect pregnancy rates (Ryan et al., 1994). Similarly, Mee et al. (1990) concluded that GnRH treatment at 1 h or

PAGE 31

19 12 to 16 h after first detected es trus did not improve pregnancy rates at first service. Mee et al. (1990) mentioned that 16 studies in th e literature suggest an overall advantage in pregnancy rate of 6 percentage points ( 53 vs. 59%) or an 11% improvement for cows receiving GnRH treatment at the time of AI or up to 6 h preceding AI. Induction of an Accessory Corpus Luteum Progesterone concentrations following ovulatio n have been positively correlated to volume of uterine secretions (Garrett et al., 1988a) conceptus development (Garrett et al., 1988a; Mann et al., 1996), the em bryos ability to secrete IFN(Kerbler et al., 1997; Mann et al., 1998), embryo viability for subse quent survival (Stronge et al., 2005), and perhaps most importantly conception rates (H ansel, 1981; Fonseca et al., 1983; Shilton et al., 1990; Larson et al., 1997). One possibl e approach to incr easing progesterone secretion has been to induce formation of an accessory CL by administering GnRH or hCG, LH or their analogues at a time when the first wave dominant follicle is present after ovulation (metestrus) (Rajamahendran and Sianangama, 1992; Schmitt et al., 1996b; Santos et al., 2001). Santos et al. (2001) reported that hCG treatment on d 5 of a synchronized estrous cycle induced an accessory CL in 86.2% of treated cows, increased plasma progesterone by 5 ng/ml, and increase d conception rates on day 28 from 38.7% to 45.8% and on day 90 of pregnancy from 31.9% to 38.4%. Lactating dairy cows treated with GnRH on d 5 (Willard et al., 2003) and hCG on day 7 (Rajamahendran and Sianangama, 1992) or day 4 in heifers (Breue l et al., 1989) reported successful accessory CL formation and an increase in co nception rates and pregnancy rate. Besides stimulating luteal tissue formati on, treatment of cows to induce ovulation of the first wave dominant follicle with GnRH or GnRH analogues also reprograms follicular growth to increase the proportion of estrous cycles composed of three follicular

PAGE 32

20 waves as compared to two waves (Diaz et al ., 1998). Such an effect could reduce the probability that a large, highly estrogenic foll icle is present during the critical period of pregnancy recognition. Compared to animal s with two-wave cycles, Holstein cows (Townson et al., 2002) and beef cows (Ahmad et al., 1997) with a three-wave cycle had higher conception rates and a longer lu teal phase (Ginther et al., 1989). Progesterone Supplementation The ability of the conceptus to secrete IFNis related to its developmental progress and progesterone concen tration of the pregnant fema le (Mann et al., 1999). Low progesterone concentration in plasma as ea rly as day 6 after insemination has been implicated as a contributing factor for cows failing to conceive (Bulman and Lamming, 1978; Lukaszewska and Hansel, 1980; Kimu ra et al., 1987; Lamming and Darwash, 1995; Inskeep, 1995; Mann and Lamming, 1999; Hommeida et al., 2004). Enhanced luteolytic signals also result from subop timal progesterone concentrations after insemination (Mann and Lamming, 1995b). Anothe r approach to increase fertility of lactating dairy cows has been to directly supplement cows with progesterone. A metaanalysis of 17 studies rev ealed that progesterone suppl ementation after insemination produced an overall improvement in concep tion rate of 5% and that the timing of progesterone supplementation was a critic al factor (Mann an d Lamming, 1999). One study revealed depressed conception rates when controlled in ternal drug releasing (CIDR) devices containing progesterone were inserted in heifers on day 1 or day 2 following estrus (Van Cleef et al., 1989). In contrast, injection of progesterone (100 mg) on day 1, 2, 3, and 4 of pregnancy advanced de velopment of conceptuses to 14 days of gestation in beef cows (Garre tt et al., 1988a). These conc eptuses had incr eased length and secreted a greater array of proteins in to medium following a 24 hour culture. When

PAGE 33

21 progesterone supplementation was initiated be ginning at day 10 of pregnancy, Macmillan et al. (1991) found a slight decrease in pregnancy rate (-2.7%), Sreenan and Diskin, (1983) obtained a small increase (4.3%), a nd Robinson et al. (1989) obtained a large increase (29.3%) in pregnancy rate. Villarro el et al. (2004) found that first and second lactation repeat-breeder Hols tein cows were 3.26 times more likely to become pregnant when cows received progesterone releasing intravaginal device (PRID, 1.55g of progesterone) on day 5 through 19 post-AI. Inhibition of Luteolysis The maintenance of a functional CL de pends directly upon the intensity of embryonic signals that attenuates endometrial secretion of PGF2 Pregnancy fails if an embryo does not produce sufficient amounts of IFNor if production is delayed until after the critical time-period between days 14 and 17 when the luteolysis would otherwise occur. Intrauterine infusions of recombinant bovine IFNfrom days 14 to 24 of the estrous cycle increased lifespan of the CL a nd duration of the estrous cycle (Meyer et al., 1995). Further studies with a la rge number of cows needs to test whether this treatment increases pregnancy rates. Co-transfer of embryonic vesicles to increase trophoblastic signals has been reported to increase pregnancy rates in embryo transfer recipients (Heyman et al., 1987). Administration of IFNby intramuscular injection, which can also block luteolysis, decreased pregnancy ra tes in heifers (Barros et al., 1992) because IFNhas several adverse actions such as cau sing hyperthermia (Newton et al., 1990). Administration of a prostanoid synthesis inhibitor could suppress the luteolytic stimulus in early pregnancy. Injection of flunixin meglumine (a pr ostaglandin synthesis inhibitor) neutralized oxytocin-induced PGF2 release, reduced the frequency of short

PAGE 34

22 cycles, and increased pregnancy rate from 33.3% in oxytocin challenged cows to 80% in oxytocin treated cows that received a flunixin meglumine injection (Lemaster et al., 1999). In another study, effects of flunixin meglumine on pregnancy rate were farm or location dependent (Purcell et al., 2005). T ogether, these results suggest that certain conceptuses are unable to inhibit uterine PGF2 secretion and that reducing prostaglandin synthesis and stimulating IFNsecretion could improve pregnancy rates. Nutritional Strategies Dairy cows reach peak production on averag e within the first 4 to 6 weeks after parturition. Unfortunately, feed and energy intake do not reach ma ximum levels until approximately 10 – 12 weeks postpartum. Th e end result is a lactating cow with insufficient nutritional requirements that enters a NEB status. As mentioned before, energy balance is defined as the difference between energy gain from feed intake minus the energy e xpenditure associated with maintenance of physiological function, growth, and milk pr oduction (Staples et al., 1990). Several studies have reported that negative ener gy status impaired repr oductive performance (Butler and Smith, 1989; Jorritsma et al., 2000 ). Different nutritional strategies to improve energy balance or alter nutrient de livery to improve re productive function are described in this section. Fat feeding to improve energy balance Fats are glyceride esters of fatty acids that can have a direct effect on the transcription of genes that encode proteins that are essential to reproductive events (Mattos et al. 2000). Dietary fats typically increase concentrations of circulating cholesterol, the precursor of progesterone (Grummer and Ca rroll, 1991). Ruminants fed

PAGE 35

23 supplemental fat often have a slight increas e in blood progesterone concentrations [see Staples et al. (1998) for review]. Hawkins et al. (1995) suggested th at the increase seen in circulating progesterone when cows are fe d supplemental fat was from a reduced rate of clearance of progesterone ra ther than an increase in progesterone synthesis. Fat supplementation has also been shown to stim ulate programmed growth of a preovulatory follicle (Lucy et al., 1993), total number of follicles (Lucy et al., 1991ab; Wehrman et al., 1991; Thomas and Williams, 1996; Beam a nd Butler, 1997; Lammoglia, 1997), and size of preovulatory follicles (Lucy et al., 1990, 1991a, 1993; Beam and Butler, 1997; Oldick et al., 1997). Garcia-Bojalil et al. (1998) reported that accumulated plasma progesterone from 0 to 50 days in milk (DIM) wa s greater, pregnancy rates im proved, and energy status did not change when cows were fed diets of 2.2% calcium salts of fatty acids (CSFA) compared to non fat-supplemented cows. Sim ilarly, Scott et al. (1995) fed CSFA at 0 or 450 g/d from 1 to 180 or 200 DIM and reporte d a tendency for CSFA to increase the proportion of cows exhibiting standing estr us (71.4% vs. 65.6) and a reduction in the proportion of cows with inactive ovaries. Other studies have also found a beneficial effect of feeding supplemental fats on fertility of lactating cows (E rickson et al., 1992; Sklan et al., 1994) while some studies have found no beneficial effect. Although fer tility results are inconsistent when cows were evaluated after being fed supplemental fat, Staples et al. (1998) suggested that positive effects (17 percentage unit improvement) are more often reported. When first AI service and conception or pregnancy rate data was examined, ten studies (Schneider et al., 1988; Bruckental et al., 1989; Sklan et al., 1989; Armstrong et al., 1990; Ferguson et

PAGE 36

24 al., 1990; Sklan et al., 1991; Ga rcia-Bojalil, 1993; Scott et al., 1995; Burke et al., 1996; Son et al., 1996) report an improvement (P < 0.10) while two studies (Erickson et al., 1992; Sklan et al., 1994) revealed a strong ne gative influence accompanied by a large increase in milk production. Among studies that reported an improvement (Armstrong et al., 1990; Ferguson et al., 1990; Sklan et al., 1991), a reduced number of services per conception by feeding a fat supplemen ted diet occurred as well. Dietary fats could favor reproductive processes through actions related to energy balance or through specific actions of indivi dual fatty acids on tissue function. Mattos et al (2000) has suggested that altered uterine and ovarian function can be mediated through specific fatty acid precursors in the diet to allow increased steroid and/or eicosanoid secretion. There are many examples of effect s of feeding diets high in specific fatty acids. Linoleic acid supplemented in the diet prepartum can stimulate arachidonic acid synthesis and lead to higher concentrations of the series 2 prostagla ndins (Thatcher et al., 1994). It is speculated that linolenic acid may compete with arachidonic acid for binding sites of a key enzyme, cyclooxygenase 2 (PGHS-2 ), which is necessary for the synthesis of PGF2 (Mattos et al., 2000; 2004). Supplementation of the diet with fish m eal has been reported to reduce uterine PGF2 secretion of lactating dairy cows (Thatc her et al., 1997). Fish meal contains relatively high concentrations of two polyunsat urated fatty acids of the n-3 family, EPA (eicosapentaenoic acid) and DHA (docosahexaen oic acid). Concentrations of EPA and DHA in fish oil have been reported to be 10.8 and 11.1% of total fatty acids (Donovan et al., 2000). EPA and DHA can inhibit secretion of PGF2 in different cell culture systems (Levine and Worth, 1984; Achard et al., 1997) including bovine endometrial cells

PAGE 37

25 (Mattos et al., 2001). Using fish meal to re place soybean meal as a source of protein, Bruckental et al. (1989) a nd Armstrong et al. (1990) re ported higher pregnancy and conception rates. These results suggest that high concentrations of EPA and DHA in the diet can reduce PGF2 endometrial secretion and aid in es tablishment of pregnancy rates. Administration of antioxidants Reactive oxygen species are a possible s ource of infertility because ovarian steroidogenic tissue (Carlson et al., 1993; Margolin et al., 199 2), spermatozoa (Rivlin et al., 2004), and preimplantation embryos (Fujit ani et al., 1997) become compromised as a consequence of free radical damage. Vitamin E (i.e., -tocopherol) and -carotene are major antioxidants present in plasma membranes of cells (Wang and Quinn, 1999; 2000). Treatment of cows with vitamin E and selenium can increase the rate of uterine involution in cows with metritis (Harrison et al., 1986) and improve fe rtilization rates in ewes (Segerson and Ganapathy, 1980) and co ws (Segerson et al., 1977). In general, however, treatment of lactating cows with vitamin E alone, through feeding or injection, had little or no benefits on postpartum cows (Kappel et al., 1984; Stowe et al., 1988; Archiga et al., 1998a; Paul a-Lopes et al., 2003). -carotene is another cellular antioxidant and is thought to be present at the interior of membranes or lipoproteins (Niki et al ., 1995). Cows fed diets deficient in -carotene had lower amounts of progesterone in the CL (Ahlswede and Lotthammer, 1978). In spite of this, its effect on fertility is cont roversial. Some author s report benefits of feeding supplemental -carotene (Ahlswede and Lotthammer, 1978; Rakes et al., 1985; Archiga et al., 1998b) whereas others do not (Wang et al., 1982; Akordor et al., 1986). There was no strong relationship be tween serum concentrations of -carotene and fertility

PAGE 38

26 in dairy cattle (Gossen et al., 2004; Gossen a nd Hoedemaker, 2005). Injection of vitamin A, a metabolite of -carotene, resulted in an increase in the number of recovered blastocysts from superovulated cows (Shaw et al., 1995). Crossbreeding Two bulls (Chief and Elevation) make up about 30% of the gene pool of U.S. Holsteins (Hansen et al., 2005). As mentione d previously, inbreedi ng coefficients are rising in American dairy cat tle (Short et al., 1992; Wigga ns et al., 1995; Young et al., 1996; Hansen, 2000; Wall et al., 2005) and th ere is some evidence that this has contributed to the decline in fertility seen in dairy cat tle (Thompson et al., 2000ab; Alvarez et al., 2005; Wall et al., 2005). Crossbreeding represents a strategy for preventing effects of inbreeding especially if the milk yield of crossbreds can approach that of Holstein cattle. A study in Canada revealed that some groups of crossbred cattle were equivalent to Holstein controls in lifetime net profit (McA llister et al., 1994). Hansen et al. (2005) conducted a study using seven large dairies in California to compar e characteristics of several crossbred animals (Normande-H olstein, Montebeliarde-Holstein, and Scandinavian Red-Holstein) versus Holsteins. Milk production as we ll as fat and protein production during the first 150 DIM among firs t lactation cows was not significantly different among breed types. Holsteins produced an average of 29.9 kg, followed by Scandinavian Red-Holstein with 29.7 kg, Montebeliarde-Holstein with 28.8 kg, and Normande-Holstein with 26.5 kg. Calving diffi culty and stillbirths were reduced in crossbred animals. Survival rates indicate that purebred animals left these dairies sooner. The first service conception rate was 22% for Holsteins compared to 30 35% for crossbreds. There were also significantly fewer days open for crossbred cows. Thus,

PAGE 39

27 crossbreeding offers some promise for enha ncing fertility. One unanswered question is the optimal type of mating scheme for the cr ossbred animals themselves and whether the resultant loss of heterosis in the F2 animals will reduce any advantage over purebred cows. Embryo Transfer The concept of using embryo transfer (ET) as a tool to increase pregnancy rates is based on the observation that disruptive even ts such as anovulation, ovulation of oocytes with low developmental competence, compromised oviductal transport or uterine environment, and insemination errors or dama ged spermatozoa all occur before the time when embryos are ordinarily transferred (d ay 6 8 after estrus) (Hansen and Block, 2004). Selection of morula and blastocyst stag e embryos for transfer offers the chance to avoid pregnancy failure associated with the early stages of embryonic development (day 0 8 after estrus). It has been proposed that during absence of heat stress, pregnancy rates following embryo transfer as compared to AI in lact ating cows are not optimal (Putney et al., 1989b; Drost et al., 1994; Ambrose et al., 1997 ). However, ET may become a more effective strategy to increase pr egnancy rates as compared to AI in lactating cows during periods of heat stress, and the magnitude of the increased temperature does not seem to influence overall success following transfer (Hansen and Archiga, 1999). As embryos advance in their development, the effect s of elevated temperatures become less significant because embryos become more resist ant to the deleterious effects of elevated temperatures (Ealy et al., 1992; Ealy and Hansen, 1994; Ea ly et al., 1995; Edwards and Hansen, 1997; Rivera and Hans en, 2001). As a result, pregnancy rates following ET

PAGE 40

28 during heat stress are higher th an pregnancy rates to AI (Put ney et al., 1989b; Ambrose et al., 1999; Al-Katanani et al., 2002a) although no t in the absence of heat stress. One potential constraint for embryo transfer in lactating cows is the short duration of estrus and lack of intense mounting activ ity seen in dairy cows (Dransfield et al., 1998). This phenomenon is exacerbated by heat stress (Nebel et al., 1997) and will limit the number of embryos transferre d in lactating cows in a pr ogram that is dependent upon estrus detection. The first report of a timed embryo transfer (TET) protocol, where ovulation was synchronized using an Ovsync h protocol, was by Ambrose et al. (1999) who evaluated the efficiency of TET using either fresh or frozen-thawed in vitro produced (IVP) embryos and TAI under heat st ress conditions. Pregnancy rates in cows that received a fresh IVP embryo were high er compared to cows in the TAI group. Limitations to Optimal Pregnancy Rates Using IVP TET For ET to replace AI on a wide scale in commercial herds ET must become an economical breeding alternative and embryos must be inexpensive to produce (Hansen and Block et al., 2004). Supe rovulation provides the best source of embryos while the most likely inexpensive source of embryos will be produced from slaughterhouse oocytes by IVP since superovulation is costly and requires intens ive management and careful synchronization of the donor cows. Although embryos produced using IVP sy stems are relativel y inexpensive as compared to embryos produced by superovula tion, pregnancy rates achieved following transfer of an IVP embryo are often less than what is obtain ed following transfer of an embryo produced by superovulation. For example, Hasler (2003) reported a 36.7% pregnancy rate for in vitro derived embryos vs. 54.8% for in vivo embryos. The reason for the poor survival of IVP embryos is not known. However, IVP embryos are different

PAGE 41

29 from in vivo embryo in terms of morphology (Massip et al., 1995; Cr osier et al., 2001; Rizos et al., 2002), gene e xpression (Bertolini et al ., 2002a; Lazzari et al., 2002; Lonergan et al., 2003), metabolism (Krisher et al., 1999; Khurana and Niemann, 2000b) and chromosomal abnormalities (Iwasaki et al., 1992; Viuff et al., 2000). One or more of these alterations likely contribu tes to the poor embryo survival after transfer. Calves born as the result of in vitro production are also more likely to experience developmental defects (Hasler et al., 2003; Farin et al., 2006). One possible strategy for increas ing pregnancy rates is to transfer two embryos into the uterine horn ipsilateral to the CL. Th is approach is based on the idea that the likelihood is increased that th e cow receives at least one em bryo competent for sustained development. In addition, the transfer of tw o embryos into the ipsilateral uterine horn to the CL is likely to increase the amounts of IFNand other embryo-derived signaling molecules in the uterus needed to maintain pregnancy and prevent luteolysis. Co-transfer of embryonic vesicles to incr ease trophoblastic signals has been reported to increase pregnancy rates in ET recipi ents (Heyman et al., 1987). In a recent study, there was a tendency for higher calving rates fo r recipients that received two embryos in the uterine horn ipsila teral to the CL as compared to recipients that received one embryo (Ber tolini et al., 2002a). The requir ement for the antiluteolytic signal in cattle to be locally administered (del Campo et al., 1977, 1983) means that one should expect pregnancy rates to be higher in cows that receive d two embryos in the same uterine horn (unilateral transfer) th an for cows that received two embryos distributed in both uterine hor ns (bilateral transfer). Th e opposite was true for heifers (Anderson et al., 1979). In other studies, transfer of embryos to create two pregnancies in

PAGE 42

30 the uterine horn ipsilateral to the CL has pr oduced a similar pregnancy rate as bilateral twins and single pregnancies (Sreenan and Diskin, 1989; Reichenbach et al., 1992) or reduced pregnancy rate as compared to bilateral transfer (Rowson et al., 1971). Cryopreservation of IVP Embryos An additional limitation to the widespread use of IVP embryos in cattle is their poor survival following cryopreservation. Hasler et al. (1995), Ambros e et al., (1999) and Al-Katanani et al. (2002a) indi cated that IVP embryos do not survive freezing as well as embryos produced in vivo based on pregnancy rates following transfer as compared to non-frozen embryos. In vitro survival rate s following thawing (Po llard and Leibo, 1993; Enright et al., 2000; Khurana and Niemann, 2000 a; Diez et al., 2001; Guyader-Joly et al., 1999) and pregnancy rates following thawing and transfer (Hasler et al., 1995; Agca et al., 1998; Ambrose et al., 1999; Al-Katanani et al., 2002a) are consistently lower for IVP embryos as compared to embryos produced in vivo by superovulation. Among the metabolic changes associated with IVP embryos linked to poor freezability is an increase in lipid conten t (Abe et al., 1999; Rizos et al., 2002). Mechanical delipidation (Tominaga et al ., 2000; Diez et al., 2001) and addition of inhibitors of fatty acid synthesis (De la Torre-Sanchez et al., 2005) can improve embryo survival following cryopreserv ation. Hatching rates were hi gher for delipidated embryos compared to controls when day 7 blastocy sts were frozen (Murakami et al., 1998), but pregnancy rates after the tran sfer of delipidated embryos was 10.5% compared to 22% for control embryos (Diez et al., 2001). Although delipidated em bryos can survive freezing conditions when tested in vitr o, special consideration must be taken since these embryos do not reflect higher pregnanc ies and remain less viable than control embryos.

PAGE 43

31 Manipulating the cryopreservation process to minimize damage to the embryo has also been considered. Of most promise ar e procedures based on vitrification, which is defined as “the solidification of a solu tion (glass formation) brought about not by crystallization but by extreme elevation in vi scosity during cooling” (Fahy et al., 1984). Vitrification depends on ra pid cooling and thawing of embryos while using high concentrations of cryoprotectants associ ated with elevated cooling rates (~2500oC/min, Palasz and Mapletoft, 1996). Although vitrif ication does not elimin ate toxic effects of cryoprotectants and osmotic damage, the rapi d cooling has been reported to decrease chilling injury and prevent damage associat ed with high lipid content (Dobrinsky, 1996; Martino et al., 1996ab). In vi tro survival rates following th e thawing of vitrified IVP embryos was either equal (Van-Wagtendonk et al., 1995) or superior to embryos frozen conventionally (Dinnys et al., 1995; Agca et al., 1998; O’Kearney-Fl ynn et al., 1998). Sensitivity of in vivo derived embryos to cryopreservation is much less and the complex environment where the embryo develops is key. It has been reported that embryos cultured in the sheep oviduct (26%) compared to synthetic oviductal fluid in culture systems (7%) were better able to to lerate freezing conditions. Embryos cultured in Buffalo rat liver cells or oviductal cells were more resistant to freezing as well as compared to embryos not subjected to co -culture (Massip et al., 1993; Leibo and Loskutoff, 1993; Te rvit et al., 1994). Summary and Objectives of the Thesis There has been a precipitous decline in fe rtility of dairy cows over the last 10-40 years and heat stress is associated with infert ility in lactating dairy cows. To characterize events associated with infertility is importa nt and the purpose of th e present series of experiments described in this thesis was to evaluate strategies that help overcome

PAGE 44

32 reproductive failure. Improving reproductive functi on in dairy cattle is of major interest and experiments were designed to 1) evaluate strategies for enhancing fertility after AI using GnRH treatment and 2) further deve lop ET using IVP embryos as a tool for increasing fertility by testing whether pregna ncy rate could be im proved by transfer of twin embryos and whether the developmental competence of embryos after cryopreservation could be improved. Figure 1-1. Rolling herd average (RHA, kg milk per lactation) calving interval (CI), and services per conception (SPC) for 143 da iry herds continuously enrolled in the Raleigh DHIA record system from 1970 to 1999 (Lucy, 2001).

PAGE 45

33 Figure 1-2. Temporal changes in first servi ce pregnancy rate and annual average milk production from high-producing Holstein-Fri esian dairy herds in north-eastern Spain. Data for pregnancy rate were r ecorded in the cool (October April months) and warm season (May-September months). Data were drawn by P.J. Hansen (unpublished) based on da ta of Lopez Gatius (2003). Milk yield (kg) 7500 8000 8500 9000 9500 10000 10500 1 9 9 1 1 9 9 2 1 9 9 3 1 9 9 4 1 9 9 5 1 9 9 6 1 9 9 7 1 9 9 8 1 9 9 9 2 0 0 0 First service pregnancy rate, (%) 15 20 25 30 35 40 45 50 Warm seaso n Milk y iel d Cool season Warm Season Milk Yield

PAGE 46

34 CHAPTER 2 EFFECTIVENESS OF ADMINISTRATI ON OF GONADOTRO PIN RELEASING HORMONE AT DAY 11, 14 OR 15 AF TER ANTICIPATED OVULATION FOR INCREASING FERTILITY OF LACT ATING DAIRY CO WS AND NONLACTATING HEIFERS Introduction One of the approaches proposed to improve fertility in cattle is administration of GnRH or GnRH analogues at day 11-15 after estr us. Injection of GnRH at this time can lead to decreased estrogen secretion (R ettmer et al., 1992a; Mann and Lamming, 1995a) in an action that likely involves luteinizati on of the dominant follicle (Thatcher et al., 1989; Rettmer et al., 1992a; Ry an et al., 1994). In some cases, extended estrous cycle length (Lynch et al., 1999) and in creased progesterone secreti on also results (Rettmer et al., 1992a; Stevenson et al., 1993; Ryan et al., 1994; Willard et al., 2003). Improvement of fertility has been seen by administrati on of GnRH or its analogues at day 11-14 in nulliparous beef heifers (Rettmer et al., 1992b) and lactating dairy cows (Macmillan et al., 1986; Lajili et al., 1991; Sheldon et al., 1993; Drew and Peters, 1994; Willard et al., 2003; Lpez-Gatius et al., 2005a). In contrast to these positive results, there was no favorable effect of similar treatments of Gn RH or GnRH analogues on pregnancy rates in other studies (Jubb et al., 1990; Stevenson et al., 1993; Ryan et al., 1994; Bartolome et al., 2005). In a meta-analysis of published result s, Peters et al. (2000) concluded that the overall effect of GnRH administration betw eendDay 11 and 14 after anticipated ovulation was positive, but that results were not consistent between studies.

PAGE 47

35 It is possible that GnRH treatment is mo re effective at increasing pregnancy rate per insemination during periods of heat stress than in cool weather because circulating concentrations of progesterone can be re duced in cows subjected to heat stress (Wolfenson et al., 2000). In addition, the anti-luteolytic process may be compromised because heat stress can decrease growth of the filamentous stage conceptus (Biggers et al., 1987) and increase uterine prostaglandin-F2 secretion from the uterus (Wolfenson et al., 1993). Beneficial effect s of GnRH treatment at da y 11-12 after insemination on fertility have been observed in lactating dairy cows during heat stress (Willard et al., 2003; Lpez-Gatius et al., 2005a). The purpose of the present series of experiments was to evaluate the effectiveness of GnRH treatment at either day 11, 14 or 15 after anticipated ovulation for improving fertility of lactating cows and heifers and determine whether the beneficial effect of GnRH was greater during summer than winter. Materials and Methods Experiment 1 GnRH Administration at Day 11 after Anticipated Ovulation in Heifers Subjected to Timed Artificial Insemination during Heat Stress The experiment was conducted at a commer cial dairy located in Trenton, Florida (29o37’ N 82o49’ W) from July to September, 2003 using 149 Holstein heifers. The heifers ranged in age from 13-23 mo (mean =539 d, SD=76) and ranged in weight from 316 to 448 kg (mean=360 kg, SD=32). Heifers we re maintained on grass pasture with supplemental grass hay. Heifers were randomly allocated to one of f our treatments in a 2 x 2 factorial design with main effects of timing of insemi nation (protocol A vs B) and treatment (vehicle vs GnRH). The experime nt was replicated twi ce with between 70 and 79 heifers per replicate. Heifers were subjected to timed artificial insemination (TAI) based on a protocol published previously (Mar tinez et al., 2002ab). On Day -10 of the

PAGE 48

36 protocol (Day 0 equals the day of anticipated ovulation), heifer s received 100 g (i.m.) of GnRH (Fertagyl, equivalent to 50 g /ml gonadorelin diaecetate tetrahydrate; (Intervet Inc. Millsboro, DE) and an unused intravag inal progesterone-rel easing device insert (EAZI-BREED CIDR insert, 1.38 g of progesterone, Pf izer Animal Health, New York, NY, USA). At Day -3, CIDR devices were re moved and 25 mg (i.m.) of prostaglandin F2 (PGF2 ; 5 ml Lutalyse, Pfizer Animal Health, New York, NY, USA) was administered. A second 100 g GnRH injec tion was given 48 h afte r CIDR withdrawal (Day -1). Regardless of estrus behavior, heifers in protocol A were inseminated 24 h after the second GnRH injection (d 0) and heif ers in protocol B were inseminated at the same time as the second GnRH injecti on (d -1). Two individuals conducted all inseminations and semen from one sire was used for all heifers. Heifers from each synchronization treatment protocol were random ly allocated to receive either 100 g of GnRH, (i.m.) or an equivalent volume (2 ml ) of vehicle (9 mg/ml of benzyl alcohol and 7.47 mg/ml of sodium chloride in water) at Day 11 after anticipated ovulation. On the day of insemination and on Day 11 after anticipated ovulation, a 10-ml blood sample was collected via coccygeal or jugular venipuncture into heparinized tubes (Becton Dickinson, Franklin Lakes, NJ) to measure the proportion of heifers successfully synchronized. An animal was considered s ynchronized if progester one concentrations were lower than 1 ng/ml on the day of insemination and greater than 1 ng/ml on Day 11 after anticipated ovulation. A third blood sample was collected in a subset of 76 heifers at Day 15 after anticipated ovulation (i.e., 4 d af ter the injection of Gn RH or vehicle) to determine the effect of GnRH treatment on serum concentrations of progesterone. Pregnancy was diagnosed by palpation per rectum at Day 44-51 after insemination.

PAGE 49

37 Blood samples were stored on ice (~2-4 h) until centrifugatio n at 2,000 x g for 20 min at 4 oC to obtain plasma. Plas ma was stored at -20 oC until assayed for progesterone concentrations using a progesterone radioi mmunoassay kit (Coat-a-Count; Diagnostic Products Corp., Los Angeles, CA). The sens itivity of the assay was 0.1 ng/ml and the intrassay and interassay CV were each 6%. Experiment 2 GnRH Administration at Day 11 after Anticipated Ovulation in Lactating Cows Subjected to Timed Artificial Insemination This study took place at the University of Florida Dairy Research Unit (Hague, Florida; 29o46’ N 82o25’ W). A total of 244 primiparous and multiparous lactating Holstein cows housed in freestall barns equi pped with a fan–and-sprinkler system were used. Cows were fed a total mixed ration (TMR) to meet or exceed requirements recommended for lactating dairy cows, were milked three times a day, and received bovine somatotropin (Posilac, Monsanto Corp., St. Louis, MO) according to manufacturer’s recommendation. Cows were subjected to the OvSynch TAI program (Schmitt et al., 1996a; Pursley et al., 1998); 100 g (i.m.) GnRH (Fertagyl equivalent to 50 g /ml gonadorelin diaecetate tetrahydrate, Intervet, Millsboro, DE) was injected at Day 0 of the protocol, 25 mg (i.m.) PGF2 (5 ml of Lutalyse, Pfizer Animal Health, New York, NY, USA) was given at Day 7, 100 g (i .m.), GnRH was again injected, i.m., at Day 9, and cows were inseminated 16 h later (t he day of anticipated ovulation). At the time of insemination (from January September, 2004), 244 cows were between 76 and 594 days in milk (DIM; mean= 176, SD= 114). Multiple individuals conducted inseminations (n=7) and multiple AI sires were used (n=45). Cows were randomly assigned within pair to receive 100 g (i.m.) GnRH or an equivalent volume (2 ml) of vehicle (9 mg/ml benzyl alcohol and 7.47 mg/ml sodium

PAGE 50

38 chloride in water) at Day 11 after anticipat ed ovulation (i.e., 11 d after insemination). Rectal temperature was record ed in a subset of cows (n =134) on the afternoon of Day 11 after TAI at 1500 – 1600 h. Pregnancy was diag nosed by rectal palpation at ~Day 46 after insemination. Experiment 3 GnRH Administration at Day 14 after Anticipated Ovulation in Lactating Cows Subjected to Timed Artificial Insemination This study was conducted at two different locations using lactating Holsteins. Farm 1 was the University of Florida Dairy Research Unit at Hague, Florida while farm 2 was a commercial dairy in Chiefland, Florida (29o30’ N 82o52’ W). Cows from farm 1 (n=307) were inseminated from February November 2004 and cows in farm 2 (n=170) were inseminated from June October 2004. At both farms, primiparous and multiparous cows were used. At farm 1, 307 cows were TAI between 76 – 590 DIM (mean= 187, SD= 102). Multiple individuals conducted in seminations (n=7) and multiple AI sires were used (n=42). At farm 2, 170 cows were used for first service after calving using seven different sires and one inseminator. The TAI protocol was designed to achieve insemination at 60 + 3 d in milk. Cows in both farms were housed in freestall barns equipped with fans and sprinklers, were fed a TMR, were milked three times a day, and received Posilac (Monstanto, St. Louis, MO) according to manufacturer’s directions. Cows in farm 1 were subjected to an OvSynch protocol as described for Experiment 2. Cows for farm 2 were subjecte d to a TAI protocol that incorporated a presynchronization with PGF2 (Moreira et al., 2001) a nd the CIDR-Synch ovulation synchronization protocol (Portaluppi a nd Stevenson, 2005). Cows received two injections of 25 mg PGF2 (i.m.) (Lutalyse) 14 d ap art starting on Day 21-27 DIM. Twelve days after the second PGF2 injection, a timed ovulation synchronization protocol

PAGE 51

39 was initiated. Cows received 100 g (i.m.) GnRH (2 ml of Cystorelin; Merial Limited, Iselin, NJ, USA) and an unused EAZI-BREED CIDR intravaginal progesteronereleasing device insert. Seven days later, CIDR devices were removed and 25 mg (i.m.) PGF2 was given. Cows received a second 100 g (i.m.) injection of GnRH at 72 h after CIDR withdrawal. Estrus was detected us ing tail chalk or KaMar estrus detection patches (KAMAR Inc., Steamboat Springs, CO, US A). Cows observed in estrus at 24 or 48 h after CIDR removal were inseminated at estrus. Cows not observed in estrus were inseminated at 72 h after CIDR withdrawal. Ovulation was anticip ated to occur 72 h after CIDR withdrawal. All an imals received the GnRH inj ection at 72 h regardless of estrus behavior. Cows were also randomly a ssigned within pair to receive either 100 g (i.m.) GnRH (2 ml of Cystorelin; Merial Limited, Iselin, NJ USA), or vehicle (as for experiment 2) at 14 d after anticipated ovul ation. Pregnancy was diagnosed by rectal palpation at ~Day 45 after insemination. Rectal temperature was recorded in a subset of 100 cows in Farm 1 and 39 cows in Farm 2 at 1500 h of Day 14 after anticipated ovulation. Experiment 4 GnRH Administration at Day 14 after Anticipated Ovulation in Lactating Cows Subjected to Timed Artifi cial Insemination Du ring Heat Stress This study took place at the University of Florida Dairy Research Unit with inseminations in April to June, 2005. A total of 137 primiparous and multiparous lactating Holstein cows ranging in DIM from 78 to 566 d (mean= 185, SD= 110) were subjected to an OvSynch protoc ol as described for Experime nt 2. Multiple individuals conducted inseminations (n=4) and multip le AI sires were used (n=22). Cows were randomly assigned within pair to receive 100 g (i.m.) GnRH or an equivalent volume (2 ml) of vehicle (9 mg/ml benzyl alcohol and 7.47 mg/ml sodium

PAGE 52

40 chloride in water) at Day 14 after anticipat ed ovulation (i.e., 14 d after insemination). Pregnancy was diagnosed by rectal palp ation at ~Day 46 after insemination. Experiment 5 GnRH Administration at Day 14 or Day 15 after Detected Estrus This study took place at a commercial dair y in Chiefland, Florida. A total of 296 primiparous and multiparous lactating Holstein cows inseminated at detected estrus were used. Cows were inseminated from April – August, 2005. At the time of insemination, cows were between 51 and 235 DIM (mean= 122, SD= 40). Estrus was detected using tail chalk or KaMar estrus detec tion patches (KAMAR Inc., Steamboat Springs, CO, USA). Estrus de tection patches were visually monitored twice (morning and afternoon) daily by the insemi nator. When cows were first diagnosed in estrus in the afternoon, insemination was performed the next morning. When estrus was first detected in the morning, cows were inseminated at that time. Cows were bred by one inseminator and 31 different sires use d. Every other day of the experiment, cows were selected to receive inje ctions at Day 14 or 15 after in semination. Within each day, cows were randomly assigned within a pair to receive 100 g (i.m.) GnRH or an equivalent volume (2 ml) of vehicle (9 mg/ml benzyl alcohol and 7.47 mg/ml sodium chloride in water). Pregnancy was diagnos ed by rectal palpation at ~Day 45 after insemination. Statistical Analysis Data on pregnancy rate were analyzed by logistic regression with the LOGISTIC and GENMOD procedures of SAS (SAS fo r Windows, Release 8.02; SAS Inst., Inc., Cary, NC). For the LOGISTIC procedure, a ba ckward stepwise logistic model was used. Variables were continuously removed from th e model by the Wald statistic criterion if the significance was greater than 0.20. The Wald 2 statistic was used to determine the

PAGE 53

41 significance of each main effect that remained in the reduced model. The adjusted odds ratio (AOR) estimates and the 95% Wald conf idence intervals from logistic regression were obtained for each variable that remained in the final statistical model following the backward elimination. Data were also analyzed by PROC GENMOD and P values for significant treatment effects are reported from this analysis. The full mathematical model for experiment 1 included main effects of in seminator, treatment, protocol, replicate, replicate x protocol, replicate x treatment, re plicate x inseminator, protocol x treatment, protocol x inseminator, treatment x inseminator. The full mathematical model for experiment 2 included the effects of season of insemination (January to March vs April to September), treatment, and season x treatment. For experiment 3, the full mathematical model included the effects of farm, treatme nt, season of insemination (warm vs cool season; farm 1 = October to March vs April to September; farm 2 = June to September vs October to November), and season x treatment, season x farm, and treatment x farm. In addition, a subset of data composed of cows from farm 2 only was analyzed where the additional factor of estrus detection (yes or no) was included in the model. For experiment 4, the full mathematical model incl uded the effects of treatment, month of insemination, parity (1 vs ot hers), sire, DIM at insemina tion class (<150 d vs > 150 d), parity x treatment, DIM class x treatment a nd month x treatment. For experiment 5, the full mathematical model included the effects of treatment, season of insemination (April and May vs June to August), parity (1 vs > 1) number of services (1, 2 and >2), DIM at insemination class (<150 d vs > 150 d) and interactions of main effects with treatment. Since interactions were not si gnificant, data were reanaly zed with main effects only.

PAGE 54

42 Data on rectal temperatures were analyzed by least-squares analysis of variance using the GLM procedure of SAS. The m odel included effects of season (Exp.2) or season, farm and farm x season (Exp. 3). A meta-analysis was performed using Mant el-Haenszel procedures available using software downloaded from http://www.pitt.edu/~super1 /lecture/lec1171/index.htm Three analyses were performed – using all experiments, experiments with GnRH treatment at Day 11, and experiments with GnRH treatment at Day 14 or 15. Results Experiment 1 GnRH Administration at Day 11 after Anticipated Ovulation in Heifers Subjected to Timed Artificial Insemination During Heat Stress Based on progesterone concentrations measur ed at insemination and at Day 11 after anticipated ovulation, estrous cycles of 137/149 (92%) of th e heifers were successfully synchronized. Pregnancy rate was not signi ficantly affected by GnRH treatment or insemination protocol. This is true whether all heifers were considered (Table 1) or only those successfully synchronized (results not shown). There was also no effect (P > 0.10) of GnRH treatment at Day 11 on concentra tions of plasma progesterone on Day 15. Values were 3.5 0.19 ng/ml for heifers receiving vehicle and 3.6 0.19 ng/ml for heifers receiving GnRH. Experiment 2 GnRH Administration at Day 11 after Anticipated Ovulation in Lactating Cows Subjected to Timed Artificial Insemination Treatment with GnRH did not significantly (P > 0.10) affect pregnancy rate per insemination (Table 2). This was true for inse minations in both cool seasons (January to March) and warm season (April to September) (results not shown). There was also no significant difference in pregna ncy rate between seasons.

PAGE 55

43 Rectal temperatures were higher (P < 0.001) for cows in the warm season (leastsquares means + SEM; 39.3 + 0.07 oC) than for cows in the cool season (38.9 + 0.07 oC). Experiment 3 GnRH Administration at Day 14 after Anticipated Ovulation in Lactating Cows Subjected to Timed Artificial Insemination Injection of GnRH increased pregnancy rates at both farms (treatment, P < 0.02; treatment x farm, non-significant) (Table 3) While pregnancy rates were lower in summer than winter (P < 0.05), the effect of GnRH was ap parent in both seasons and the season x treatment interaction was not significant. Cows in farm 2 were monitored for estrus. No cows were seen in estrus at 24 h after PGF2 4.7% (8/171) were detected in estr us at 48 h, 32.2% (55/171) at 72 h, and 63.1% (108/171) were not detected in estrus. Co ws in estrus at 48 h were inseminated at that time while other cows (those seen in estrus at 72 h and those not seen in estrus) were inseminated at 72 h. There was an estrus de tection class (detecte d in estrus vs not detected) x treatment interaction (P < 0.03) on pregnancy rate per insemination that reflected the fact that GnRH was effective at increasing pregnancy rate for those cows displaying estrus [3/29 (10%) for control and 12/34 (35%) for GnRH[ but had no effect for those cows not displaying estrus [7/54 ( 13%) for control and 4/54 (8%) for GnRH]. Rectal temperatures were higher (P < 0.01) for cows in the warm season (leastsquares means + SEM: 39.4 + 0.06 oC) than for cows in the cool season (39.1 + 0.11 oC) and higher (P < 0.001) for farm 2 (39.5 + 0.10 oC) than for farm 1 (39.1 + 0.07 oC), but there was no farm x season interaction. Experiment 4 GnRH Administration at Day 14 after Anticipated Ovulation in Lactating Cows Subjected to Timed Artifi cial Insemination Du ring Heat Stress Treatment with GnRH did not significantly affect pregnancy rate (Table 4). Pregnancy rate was higher (P<0.02) for cows inseminated at or before 150 DIM (30.3%,

PAGE 56

44 20/66) than for cows inseminated after 150 DIM (12.7%, 9/71). There were no other significant main effects or in teractions of GnRH treatmen t with other effects. Experiment 5 GnRH Administration at Day 14 or Day 15 after Detected Estrus Overall, pregnancy rate was higher (P< 0.0001) for cows inseminated in April and May (55/171, 32.2%) than for animals inseminated in June, July or August (12/125, 9.6%). There were, however, no other significan t main effects or interactions of GnRH treatment with other effects. Pregnancy rates were 25.6% (32/125) for cows receiving vehicle at day 14 or 15, 20.7% (19/92) for cows receiving GnRH at Day 14, and 20.3% (16/79) for cows receiving GnRH at Day 15. Overall Effectiveness of GnRH Treatme nt as Determined by Meta-Analysis When data from multiple experiments were considered together by meta-analysis, there was no significant effect of GnRH on pregnancy rate. This was the case when all experiments were consider ed (odds ratio=0.97; 95% CI=0.63, 1.50), or whether experiments with GnRH treatment on Day 11 (odds ratio=0.87; 95% CI=0.50, 1.50) or Day 14 or 15 (odds ratio=1.06; 95% CI=0.68, 1.65) were considered separately. Discussion Overall, there was no significant effect of GnRH treatment on pregnancy rate. In particular, GnRH treatment at Day 11 af ter anticipated ovulation did not improve pregnancy rate of heifers or lactating cows in any expe riment, whether animals were exposed to heat stress or not. Moreover, Gn RH did not consistently improve fertility when given at Day 14 after anticipated ovulatio n or at Days 14 or 15 after insemination. In one experiment (experiment 3), administ ration of GnRH at Day 14 after anticipated ovulation in cows subjected to TAI increased pregnancy rate of lactating cows in

PAGE 57

45 summer and winter at two locations. However, this positive effect c ould not be replicated either in lactating cows subjected to TAI or for cows inseminated at standing estrus. The variability in response to GnRH is reminiscent of the results of the metaanalysis of published studies performed by Pe ters et al. (2000) in which inconsistency between studies was noted. Variability in results could reflect either error in estimates of treatment effects because of small numbers of experimental units or variability in biological responses to GnRH. The number of animals used for the present studies varied and could have been too small in some studies to detect significan t differences or have lead to sampling errors that obscured the magnitude or direction of the treatment differences. However, meta-analysis of the entire data set, involving 1303 cows, indicated that there was no overall effect of GnRH. It is also possible that herds differ between each other or over time in the predominant biological response to GnRH treat ment. Presumably, beneficial effects of GnRH post-insemination on fertility are relate d to its actions to cause LH release. Treatment with GnRH at Day 11-15 of the es trous cycle can decrea se function of the dominant follicle (Thatcher et al., 1989; Rettmer et al., 1992 a; Ryan et al., 1994; Mann and Lamming, 1995a) and increase progester one secretion (Re ttmer et al., 1992a; Stevenson et al., 1993; Ryan et al., 1994; Willard et al., 2003). The reduction in estradiol-17 secretion caused by GnRH should dela y luteolysis and conceivably allow a slowly-developing conceptus additional time to initiate secretion of interferon. Low progesterone secretion may also compromise fertility in dairy cattle (Mann and Lamming, 1999; Lucy, 2001) and an increase in progesterone secretion caused by GnRH may facilitate embryonic development. Whether a herd responds to GnRH by undergoing

PAGE 58

46 follicular changes may depend upon the character istics of follicular growth because a follicle must reach 10 mm in diameter to ovulate in response to LH (Sartori et al., 2001). Perhaps, herds that do not respond to GnRH w ith an increase in fert ility are herds where many cows have lower follicular growth or follicular wave characteristics that do not result in sufficient follicular development at the time of injection. One example of the potential importance of follicular dynamics in determining responses to GnRH is the expected res ponse to GnRH treatment at Day 11 after anticipated ovulation. In the current studi es, injection of GnRH at Day 11 after anticipated ovulation did not incr ease pregnancy rates in either lactating Holstein cows or nulliparous heifers. For lactating cows, the absence of an effect of GnRH at Day 11 was seen in both summer and winter. This result which agrees with other studies in which injection of GnRH at Day 11 does not affect fertility (Stevenson et al., 1993; Jubb et al., 1990), is in contrast to othe r studies indicating that GnRH treatment at Day 11 can increase fertility of heifers (Rettmer et al., 1992b) and lactating cows (Sheldon and Dobson, 1993; Willard et al., 2003). One factor that could influence the effectiveness of GnRH treatment at Day 11 is the number of follicular waves that an individual animal expresses. Animals with estrous cycles characterized by three follicular waves have larger second-wave dominant follicles at Day 11 of the estrous cycl e than animals with two-wave cycles (Ginther et al., 1989; Savio et al., 1990; Ko et al., 1991) and thus the preponderance of cycle type (two-wave vs three-wave) within a herd may determine effectiveness of GnRH treatment at Day 11. There is variation from study to study in the relative frequency of three-wa ve vs two-wave cycles, at least among Holstein heifers (Ginther et al., 1989; Knopf et al., 1989; Ra jamahendran et al., 1991; Gong et al., 1993),

PAGE 59

47 and this variation is evidence for herd-to-herd variation in frequenc y of follicular wave patterns. Even in animals with three-wave follicul ar cycles, Day 11 woul d appear to not be an optimal time of the estrous cycle for us ing GnRH to cause luteinization because the second-wave dominant follicle is smaller at Da y 11 than at 14-15 in heifers (Ginther et al., 1989; Ko et al., 1991) and lactating cows (Ko et al., 1991). Results from a limited number of cows in Experiment 3 suggested that the effectiveness of GnRH at Day 14 after anticipated ovulation depends upon wh ether cows are detected in estrus. Presumably, ovulation occurred on average sooner for cows in estrus at 48 and 72 h after prostaglandin than for cows not detected in estrus (which contains cows that had not initiated estrus by 72 h as well as some cows in which estrus occurred by 72 h but was not detected). Among those detected in estr us, GnRH injection improved fertility from 10.3% to 35.3%. Among animals not dete cted in estrus, however, there was no difference in pregnancy rate between anim als treated with vehicle (13.0%) or GnRH (7.6%). It is likely that GnRH did not affect pregnancy rate in the cows not detected in estrus because this group included cows that were anovulatory at insemination or that were not successfully synchronized; GnRH w ould be unlikely to increase pregnancy rate in these animals. It was hypothesized that bene ficial effects of GnRH would be greater during heat stress because this condition can decrease gr owth of the filamentous stage conceptus (Biggers et al., 1987), increas e uterine prostaglandin F2 secretion from the uterus (Wolfenson et al., 1993) and reduce circul ating concentrations of progesterone (Wolfenson et al., 2000). Bene ficial effects of GnRH tr eatment at Day 11-12 after

PAGE 60

48 insemination on fertility have been observed in lactating dairy cows during heat stress (Willard et al., 2003; Lpez-Gatius et al., 2005a ). There was no evidence, however, that GnRH was more effective during the summer. In particular, the in crease in pregnancy rate caused by injection of GnRH at Day 14 during experiment 3 was similar for cows inseminated in summer and winter. In ot her experiments conducted during the summer, GnRH was without be neficial effect. In experiment 1, there were no differences in pregnancy rates for Holstein heifers inseminated either at second GnRH injection (24.4%) or 24 after GnRH (19.8%). This result is similar to results of Pursley et al. (1998) who reported little difference in pregnancy rates and no differen ces in calving rates between l actating cows inseminated at 0, 8, 16, or 24 h after the second GnRH injection of the OvSynch regimen. The pregnancy rates achieved with heifers in experiment 1 were lo w compared to other studies in which heifers rece ived a similar ovulation synchronization program (Martinez et al., 2002ab). The low fertility was not a result of delayed pubert y or unresponsiveness to the synchronization protocol because 92% of the heifers had both low progesterone concentrations during the expected pe riovulatory period a nd high progesterone concentrations at the predicted lu teal phase of the cycle. It is possible that some of these heifers classified as synchronized experienced short estrous cycles (Schmitt et al., 1996b; Moreira et al., 2000a). The experiment was co nducted during the summer and it is also possible that heat stress reduced fertility. A lthough fertility in Hols tein heifers does not always decline during the summer (Ron et al ., 1984; Badinga et al., 1985), there is one report (Donovan et al., 2003) that heifers from a dairy farm in north central Florida inseminated in summer were more than four times less likely to become pregnant to first

PAGE 61

49 insemination than heifers insemina ted during the rest of the year. It is also possible that the one sire used to inseminate all heifers was not a fertile bull. In conclusion, injection of GnRH at Day 11-15 after anticipated ovulation or insemination did not consisten tly increase pregnancy rates in heifers or lactating cows. The fact that GnRH administration was eff ective in one study indicates that such a treatment may be useful for increasing pregna ncy rate in some herd s or situations. More work will be required to describe factors that could identify which groups of cows would be most likely to benefit from GnRH treatment. Table 2-1. Descriptive statistics, adjusted odds ratio (AOR) estimates, and 95% Wald confidence intervals (CI) for effect of GnRH administrati on at Day 11 after anticipated ovulation and ovulation synchronization protocol on pregnancy rates of heifers during heat stress. 1 Data represent the number of females pregnant at Day 44-51 after insemination / total number of females inseminated. 2 Derived from PROC GENMOD. 3 Wald chi-square statistic =0.54 (N.S). 4 Wald chi-square statistic = 0.40 (N.S.) Pregnancy rate Proportion 1 % AOR95% Wald CI P -value 2 GnRH Treatment 3 GnRH 20/78 25.61.29 0.59 – 2.83 0.41 Vehicle 14/71 19.7 Protocol 4 B 20/79 25.31.34 0.61 – 2.95 0.41 A 14/70 20.0

PAGE 62

50 Table 2-2. Descriptive statistics, adjusted odds ratio (AOR) estimates, and 95% Wald confidence intervals (CI) for effect of GnRH administrati on at Day 11 after anticipated ovulation and season of insemination on pregnancy rates of lactating cows subjected to timed artificial insemination. 1Data represent the number of females pregnant at ~d 45 after insemination / total number of females inseminated. 2 Derived from PROC GENMOD. 2 Wald chi-square statistic =1.50 (N.S). 4 Wald chi-square statistic = 1.38 (N.S.) Table 2-3. Descriptive statistics, adjusted odds ratio (AOR) estimates, and 95% Wald confidence intervals (CI) for effect of GnRH administrati on at Day 14 after anticipated ovulation and season of insemination on pregnancy rates of lactating cows subjected to timed artificial insemination. 1 Data represent the number of females pregnant at ~Day 45 after insemination / total number of females inseminated. 2 Derived from PROC GENMOD. 3 Wald chi-square statistic =4.94 (P=0.026). 4 Wald chi-square statistic = 5.12 (P=0.024) Pregnancy rate Proportion 1 % AOR95% Wald CI P -value 2 GnRH Treatment 3 GnRH 26/121 21.50.66 0.37 – 1.18 0.16 Vehicle 36/123 29.3 Season 4 January – March 30/103 29.11.38 0.77 – 2.48 0.27 April September 32/141 22.7 Pregnancy rate Proportion 1 % AOR95% Wald CI P -value 2 GnRH Treatment 3 GnRH 49/241 20.31.76 1.07 – 2.89 0.02 Vehicle 30/236 12.7 Season 4 Oct, Nov, Feb, March 40/187 21.41.76 1.08 – 2.87 0.02 May September 39/290 13.5

PAGE 63

51 Table 2-4. Descriptive statistics, adjusted odds ratio (AOR) estimates, and 95% Wald confidence intervals (CI) for effect of GnRH administrati on at Day 14 after anticipated ovulation and Da ys in milk (<150 d vs > 150) at insemination on pregnancy rates of lactating cows subj ected to timed artificial insemination during heat stress. 1 Data represent the number of females pregna nt at ~Day 45 after insemination / total number of females inseminated. 2 Derived from PROC GENMOD. 3 Wald chi-square statistic =3.55 (P=0.060). 4 Wald chi-square statistic = 6.12 (P=0.013) Pregnancy rate Proportion 1 % AOR 95% Wald CI P -value 2 GnRH Treatment 3 GnRH 11/73 15.1 0.43 0.18 – 1.04 0.05 Vehicle 18/64 28.1 Days in milk at insemination 4 < 150 d 20/66 30.3 3.11 1.27 – 7.62 0.02 > 150 d 9/71 12.7

PAGE 64

52 CHAPTER 3 EFFECT OF TRANSFER OF ONE OR TWO IN VITRO-PRODUCED EMBRYOS AND POST-TRANSFER ADMINISTRATI ON OF GONADOTROPIN RELEASING HORMONE ON PREGNANCY RATES OF HEAT-STRESSED DAIRY CATTLE Introduction The in vitro produced (IVP) embryo is di fferent from the embryo produced in vivo in terms of morphology (Iwasak i et al., 1992; Massip et al., 1995; Crosier et al., 2001), gene expression (Bertolini et al., 2002a; L azzari et al., 2002; Lonergan et al., 2003), metabolism (Khurana et al., 2000b), and in cidence of chromosomal abnormalities (Iwasaki et al., 1992; Viuff et al., 2000). No t surprisingly, pre gnancy rates achieved following transfer of an IVP embryo are of ten less than what is obtained following transfer of an embryo produced by superovulatio n and calves born as the result of in vitro production are more likely to experience deve lopmental defects (Hasler et al., 2003). Problems associated with the transfer of IVP embryos have limited the realization of the potential of these embryos for enhanci ng genetic improvement and reproductive performance of lactating dairy cattle (Ru tledge, 2001; Hansen and Block et al., 2004). One method that might be useful for in creasing pregnancy ra tes in dairy cattle recipients that receive an IVP embryo is to transfer two embryos into the uterine horn ipsilateral to the CL. Such a treatment might increase pregnancy rate because the likelihood is increased that th e cow receives at least one em bryo competent for sustained development. In addition, the transfer of tw o embryos into the ipsilateral uterine horn is likely to increase the amounts of interferonand other embryonic signaling molecules in the uterus needed to maintain pregnancy and prevent luteolysis. Co-transfer of

PAGE 65

53 embryonic vesicles to increase trophoblasti c signals has been reported to increase pregnancy rates in embryo transfer recipien ts (Heyman et al., 1987). For the current experiment, both embryos were transferred in to the uterine horn ipsilateral to the CL because of the requirement for the antiluteolytic signal in cattle to be locally administered (Del Campo et al., 1977; 1983). In a recent st udy with a small number of transfers (n=10 to 28 recipients), there was a tendency for hi gher calving rate for recipients that received two embryos in the uterine horn ipsilateral to the CL as compared to recipients that received one embryo (Bertolini et al., 2002b). Anderson et al. (1979) found a tendency for pregnancy rates to be highe r in cows that received two embryos in the same uterine horn (unilateral transfer) than for cows that received two embryos distributed in both uterine horns (bilateral tran sfer); the opposite was true fo r heifers. In other studies, transfer of embryos to create tw o pregnancies in the uterine horn ipsilateral to the CL has produced a similar pregnancy rate as bilatera l twins and single pre gnancies (Sreenan and Diskin, 1989; Reichenbach et al., 1992) or reduced pregnancy rate as compared to bilateral transfer (R owson et al., 1971). Another treatment that has potential for increasing pregnancy rates in embryo transfer recipients is injec tion of GnRH at Day 11 after th e anticipated day of ovulation. Such a treatment was shown to increase pregna ncy rates in heat-stre ssed, lactating cows following insemination (Sheldon and Dobs on, 1993; Willard et al., 2003) and embryo transfer (Block et al., 2003) Treatment with GnRH or its analogues at Day 11 to12 of the estrous cycle has been reported to increase progesterone secretion (Ryan et al., 1994; Willard et al., 2003) and inhibit function of the dominant follicle (Savio et al., 1990; Ryan et al., 1994) to possibly delay luteolysis.

PAGE 66

54 The purpose of the current pair of experiments was to examine the effectiveness of unilateral transfer of twin embryos and treatment with GnRH at Day 11 after the anticipated day of ovulation for increasing pregna ncy rates in dairy catt le recipients that received IVP embryos. Experiments were performed during periods of heat stress because embryo transfer offers benefits as a method for increasing pregnancy rate as compared to AI in females subjected to heat stress (Rutledge, 2001). Materials and Methods Experiment 1 Single or Twin Transfer of IVP Embryos into Crossbred Dairy Recipients The experiment was conducted at a comm ercial dairy located in Santa Cruz, Bolivia (17o48’ S, 63o10’ W) from November – Decembe r, 2004. Data on minimum and maximum air temperatures during the experi ment collected by Servicio Nacional de Meteorologa e Hidrologa ( http://www.senamhi.gov.bo/meteorologia/ ) for Santa Cruz are presented in Figure 1. Females receivi ng embryos included 32 virgin crossbred heifers sired by Simmental, Gyr, or Brown Swiss bulls and Holstein or Holstein crossbred dams and 26 lactating, crossbred cows with the proportion of Holstein varying from 1/2 to 15/16. The heifers ranged in age from 363 to 2070 d (mean = 850 d and median = 664 d; SD = 421 d) and ranged in weight from 247 to 430 kg (mean = 310 kg and median = 288 kg; SD = 52.3 kg). Animals were maintained on grass pasture until two weeks prior to the start of the synchronization program when they also received a supplement of 6 kg/head/d of spent brewers’ grain. The cows ranged in age from 820 to 4075 d (mean = 2083 d and median = 1670 d; SD = 986 d), were maintained on grass pasture, and received 11 kg of brewers’ grains and 2 kg of a soybean-based concentrate mixture before each milking. Cows were milked two times per day and ranged from 110

PAGE 67

55 to 417 d in milk (mean =190 d and median = 170 d; SD = 75 d). Milk yield per day across all days of lactation ranged from 5.9 to 21.1 kg/d (mean = 12.5 kg/d and median = 12.6 kg/d; SD = 3.8 kg/d). Recipients were synchronized for timed em bryo transfer using a modified OvSynch protocol (Portaluppi and Steven son, 2005) with the inclusion of a controlled intravaginal drug releasing device (EAZI-BREED CIDR insert, 1.38 g of progesterone, Pfizer Animal Health, New York, NY, USA). On Day -10 (Day 0 equals the day of anticipated ovulation), females received 100 g (i.m.) of GnRH (1 ml of Profertil; Tortuga Cia. Zootcnica Agrria, So Paulo, Brazil) and an intravaginal progesterone-releasing device insert that had been used one time previous ly. On Day -3, CIDR devices were removed and females received 150 g (i.m.) of PGF2 (2 ml of Prostaglandina Tortuga, Tortuga Cia. Zootcnica Agrria). On Day 0, 100 g (i.m.) of GnRH was administered. Behavioral symptoms of estr us were monitored about 5 times each day for 3 d following CIDR removal and PGF2 injection. On Day 6 after antic ipated ovulation, all females, including those not seen in estrus, were exam ined per rectum for the presence of a CL using an Aloka 210 ultrasound unit equipped wi th a 5 MHz linear array probe (Aloka, Wallingford, CT, USA). A group of females having a CL (n=32 heifers and n=26 cows) were randomly selected within recipient type (heifers or cows) to receive one (n=31 females) or two (n=27 females) embryos on Day 7 after anticipated ovulation. For embryo transfer, an epidural block of 5 ml of lidocaine hydrochloride (2% w/v; Sparhawk Laboratories Inc., Lenexa, KS, USA) was admini stered to each recipi ent, and one or two IVP embryos were deposited in to the uterine horn ipsilatera l to the ovary containing the CL. One technician conducted all transfers.

PAGE 68

56 A total of 85 blastocysts (72 at Day 7 after insemination and 13 and Day 8 after insemination) were transferred in this expe riment. Of these, six were produced by Transova (Sioux City, IA, USA) using Holste in oocytes and a Holstein sire and were cultured in Synthetic Oviductal Fluid (SOF) medium. Embryos were shipped overnight in a portable incubator to Gainesville, FL, USA on Day 4 after insemination. Embryos were transferred to fresh microdrops of a modified SOF (Fischer-Brown et al., 2002) prepared by Specialty Me dia (Phillipsburg, NJ, USA) and cultured at 38.5oC in a humidified atmosphere of 5% O2 and 5% (v/v) CO2 (balance N2). The remainder were produced using oocytes obtained from ovaries of a variety of breeds collected at a local abattoir located at a travel distance of approximately 1.5 h from the Gainesville laboratory. Procedures, reagents, and media formulation for oocyte maturation, fertilization, and embryo culture were as previously described (Roth and Hansen, 2005) with some modifications. Cumulus-oocyte complexes were matured for approximately 22 h at 38.5C in an atmosphere of 5% (v/v ) CO2 in humidified air and then inseminated with a cocktail of Percoll-purified spermatozoa from three different bulls of various breeds. At 8 – 12 h post-insemination (hpi), putative zygotes were denuded of cumulus cells by suspension in Hepes-TALP medium (Caisson, Rexburg, ID, USA) containing 1000 units/ml hyaluronidase type IV (Sigma, St Louis, MO, USA) and vortexed in a microcentrifuge tube for 5 min. Presumptive zygotes were then placed in groups of ~30 in 50 l microdrops of KSOM-BE2 (Soto et al ., 2003) (Caisson, Rexburgh, ID, USA) at 38.5C in an atmosphere 5% (v/v) CO2 in air. Regardless of method of production, embryos greater than 16 cells in appearance were collected at 1300 h on Day 6 or 7. Embryos were placed in groups of 21 to 65 into

PAGE 69

57 2 ml cryogenic vials (Nalge Company, Roches ter, NY, USA) filled to the top with KSOM-BE2 that was pre-warmed and equilibr ated in 5% (v/v) CO2 in air. Embryos produced by Transova were kept separately from those produced using ovaries from the local abattoir. Vials containing embryos were placed in a portable incubator (Minitube of America, Verona, WI, USA) that had been pre-warmed to 39oC for 24 h prior to use. Embryos were shipped by air and arrived at Santa Cruz de la Si erra, Bolivia, at 1100 h the next day (Day 7 or 8 after in vitro insemination) and transported by ground to the farm. Embryos were transferred over a time span from 1300 h and 2000 h. One or two embryos were loaded into 0.25 cc straws in Hepes-TALP (Caisson) containing 10% (v/v) bovine steer serum (Pel-Freez, Rogers, AR, USA) and 100 M 2-mercaptoethanol (Sigma-Aldrich, St. Louis, MO, USA). Embryos were transferred to recipients that were palpated the day before and had a detectable CL. Recipien ts were randomly assigned to receive one or two embryos, and all embryos we re transferred into the ipsilateral horn to the CL. Pregnancy diagnosis was performed by rectal palpation at Day 64 and 127 posttransfer, and the number of fetuses was reco rded on Day 127. Data collected at calving included length of gestation (with the day of transfer being considered Day 7 of gestation), occurrence of dystocia (defined as needing assistance), sex, weight and viability of each calf, and o ccurrence of retained placenta (f ailure of the placenta to be expelled within 12 h after calving). Calf surviv al until Day 7 of age was also recorded. Experiment 2 Administration of GnRH on Day 11 after Anticipated Ovulation in Lactating Recipients that Received an IVP Embryo This study took place at a commercial dairy located in Bell, FL, USA (29o 45’ N 82o 51’ W) from June to October, 20 04. Data on minimum and maximum air

PAGE 70

58 temperatures and average relative humidity collected by the Florida Automated Weather Service ( http://fawn.ifas.ufl.edu ) for Alachua, FL, USA are presented in Figure 1. A total of 87 multiparous, lactating Holstein cows in la te lactation were used as recipients. Cows were fed a total mixed ration to meet or exceed requirements recommended for lactating dairy cows, milked three times a day, and received bovine somatotropin (Posilac, 500 mg sometribove zinc, Monsanto, St. Loui s, MO, USA) accordi ng to manufacturer’s directions. Cows were housed in a dry lot with access to a permanent shade structure without fans or sprinklers a nd with access to a cooling pond. Cows were prepared for embryo transfer in groups of 6 to 18; a total of 10 replicates were completed. To synchronize re cipients for timed em bryo transfer, cows received 100 g (i.m.) of GnRH (2 ml of Cystorelin; Merial Limited, Iselin, NJ, USA), on Day –10; 25 mg (i.m.) of PGF2 on Day -3; and 100 g (i.m.) of GnRH, on Day 0 (i.e., the day of anticipated ovulation). On Day 7 after anticipated ovulation, all cows were palpated per rectum for the presence of a CL. Cows that had a palpable CL received an epidural block of 5 ml of lidocaine (2%, w/v), and a si ngle embryo was transferred to the uterine horn ipsilateral to the ovary c ontaining the CL. Recipients were randomly assigned to receive 100 g (i.m) of GnRH or vehicle (9 mg/ml of benzyl alcohol and 7.47 mg/ml of sodium chloride in water) on Day 11 after anticipated ovulation. The embryos used for transfer were produ ced in the Gainesville laboratory using oocytes of various breeds and a pool of seme n from three bulls of various breeds as described for Experiment 1. A different pool of semen was used for each replicate. Presumptive zygotes were cultured in groups of ~30 in 50 l microdrops of modified SOF (Fischer-Brown et al., 2002) containing 100 ng/ml of insulinlike growth factor-1

PAGE 71

59 (Upstate Biotechnology, Lake Placid, NY, USA) Embryos were cultured at 38.5C in a humidified atmosphere of 5% (v/v) O2 and 5% (v/v) CO2 with the balance N2. On Day 7 after insemination, blastocysts were harveste d and transported to the farm in 2 ml cryogenic vials (20 to 25 embr yos/tube) filled to the top wi th pre-warmed Hepes-TALP. Tubes containing embryos were placed in a portable incubator (Minitube of America, Verona, WI, USA) that had been pre-warmed to 39oC for 24 h prior to use. Embryos were transported to the farm and loaded in 0.25 cc straws prior to transfer into recipients. Pregnancy was diagnosed by rect al palpation at Day 45 to 53 after anticipated ovulation. Statistical Analysis Categorical data were analyzed by lo gistic regression using the LOGISTIC procedure of SAS for Windows (Version 9, SAS Institute Inc., Cary, NC, USA) with a backward stepwise logistic model. Variable s were continuously removed from the model by the Wald statistic criterion if the significan ce was greater than 0.2. The full statistical model for Experiment 1 included treatment ( one embryo or two embryos), parity (cows vs heifers), estrus (observe d in estrus vs not observed) and treatment x parity on pregnancy rate, pregnancy loss, calving rate, calf mortalit y and twinning rate. The only variable in the final mathematical model fo r Experiment 2 was GnRH treatment as other effects (replicate and replicate x treatment) were not significant. The adjusted odds ratio estimates and the 95% Wald confidence inte rvals (CI) from logistic regression were obtained for each variable that remained in the final statistical model following the backward elimination. Data were also anal yzed with the GENMOD procedure of SAS to determine the significance of each effect that remained in the reduced model; P values for logistic regression analyses repo rted in the tables are derived from these analyses. Data for gestation length and calf bi rth weight were analyzed by analysis of variance using

PAGE 72

60 Proc GLM. The full statistical model incl uded the effects of treatment, parity and treatment x parity. The 2 test was used to determine whether the sex ratio of calves differed from the expected 1:1 ratio. Results Experiment 1 Single or twin transfer of IVP embryos Pregnancy and calving rates Data are summarized in Tabl e 1. At Day 64 of gestation, the pregnancy rate tended to be higher (P = 0.07) for cows than for heifers. Wh ile there were no si gnificant effects of number of embryos transferred or parity x number transferred, heifers that received two embryos tended to have lower pregnancy rates than those that received a single embryo (20% for two embryos vs 41% for one embryo) while there was no difference in pregnancy rate due to number of embryos transferred to cows (50% for two embryos vs 57% for one embryo). Pregnancy losses between Day 64 and 127 occurred in one group only – cows receiving two embryos. In that group, pregna ncy rate was 50% at Day 64 but decreased to 17% at Day 127. There was no difference in pregnancy rates at Day 127 between cows and heifers, but recipients that r eceived two embryos had lower pregnancy rates (17% for cows and 20% for heifers) than re cipients that received one embryo (57% for cows and 41% for heifers, P < 0.03). Pregnancy loss after Day 127 occurred in one female only. In particular, a cow receiving a single embryo gave birth to a st illborn calf at 251 d of gestation. Like for pregnancy rate at Day 127, there was no diffe rence in calving rate between cows and heifers, but recipients that received two embryos had lower calving rates (17% for cows

PAGE 73

61 and. 20% for heifers) than reci pients that received one em bryo (50% for cows and 41% for heifers, P < 0.03). Estrus was detected at 24, 48 or 72 h afte r prostaglandin injection in 21/32 heifers (8 at 24 h after injection and 13 at 48 h) and 19/26 cows (1 at 24 h after injection, 14 at 48 h and 4 at 72 h). While not statistically different (P=0.11), th ere was a tendency for pregnancy rates to be lower for animals not de tected in estrus. For example, pregnancy rates at Day 127 for animals receiving one embr yo was 55% (11/20) for animals in estrus vs 36% (4/11) for animals not observed in es trus. Pregnancy rates at Day 127 for animals receiving two embryos were 25% (5/20) for anim als in estrus vs 0% (0/7) for animals not observed in estrus. Characteristics of gestation, parturition, and calves Gestation length was affected by recipient type x number of embryos transferred (P<0.05; Table 2). For cows, gestation length was slightly longer fo r those receiving one embryo as compared to those receiving two embryos while the opposite was true for heifers. Two of 5 females calving that receiv ed two embryos produced twin calves. There was no significant effect of r ecipient type or number of em bryos transferred on dystocia or incidence of retained placenta (Table 2) Sex ratio (including the one stillborn calf) was in favor of males with 15 males compared to 7 female calves born (68% male; Table 3). This ratio tended to be different from the expected 1:1 ratio (P<0.10). While there were no significant differences there was a tendency for calf mortality at birth to be greater for he ifers receiving two embryos than for other groups (Table 3). None of the cows lost their calf at birth a nd only 1 of 7 heifers receiving a single embryo experienced calf death at birth. In contra st, 2 of 3 heifers receiving two embryos experienced calf loss. One heifer had twin fe tuses and both were born dead as a result of

PAGE 74

62 complications with calving. Anot her heifer gave birth to a si ngle calf that was born dead as a result of complications w ith calving. The calf from the third heifer was born alive. All calves born alive were alive 7 d later. Experiment 2 Administration of GnRH on Day 11 after Anticipated Ovulation Administration of GnRH at Day 11 afte r anticipated ovulation had no effect (P>0.10) on pregnancy rates. Recipients treated with GnRH had a pregnancy rate of 17.8% (8/45) while those recipients that re ceived placebo had a pregnancy rate of 16.7% (7/42). The odds ratio was 1.08 with 95% Wald confidence interval of 0.23 and 3.30. Discussion The purpose of the experiments described here was to examine two strategies for increasing pregnancy rates in heat-stressed da iry recipients that receive an IVP embryo. Neither approach, transferring tw o embryos into the uterine horn ipsilateral to the CL or injection of GnRH at Day 11 after anticipated ov ulation, increased pregnancy rates. Results of Experiment 1 indicated that the transfer of two embr yos into recipients led to pregnancy loss and that such loss occurred earlier for heifers than for cows. There was a distinct difference in pregnancy rate between heifers that received one or two embryos as early as Day 64 of gestation. Among cows, in contrast, there were no differences in pregnancy rate at this stage of gestation betw een recipients that received one or two embryos. By Day 127, however, cows that received two embryos experienced substantial mid-to-late fetal loss and pregna ncy rate and subsequent calving rate was lower for this group than for cows that received a single embryo. The most likely explanation for the incr eased frequency of pregnancy loss in recipients receiving two embryos is uterin e crowding, with the effects of crowding occurring sooner in gestation for nulliparous animals than for multiparous animals.

PAGE 75

63 Similar results were obtained in anothe r study (Anderson et al., 1979). In that study, calving rates and twinning rate s were similar for cow recipients regardless of whether twin transfers were performed via bilateral or unilateral placeme nt. For heifers, in contrast, calving rate and twinning rate was lower for unilateral twin transfers than for bilateral transfers. Using heifers, Rowson et al. (1971) also found lower embryonic survival rates and twinning rates for recipien ts of unilateral twin transfers than for recipients of bilateral transfers. It is evident, however, that uterine capacity can vary between herds of cattle. Thus, there were no differences in pregnancy su ccess between recipients of twin embryos placed unilaterally or bilaterally for heifers (Sreenan and Diskin 1989; Reichenbach et al., 1992) or cows (Sreenan and Diskin 1989). Si milarly, embryonic survival rate for beef cows selected for twinning was similar for those having unilateral or bilateral multiple ovulations (Echternkamp et al., 1990). In lact ating dairy cows, in contrast, the likelihood of a twin pregnancy resulting from multip le ovulation going to term was higher if ovulations occurred bilaterally than if unilateral o vulations occurred (Lpez-Gatius et al., 2005b). Perhaps, identification of the biological processes controlling uterine capacity will lead to new approaches for increasing the efficacy of producing twins in cattle. In an earlier study, administration of GnRH at Day 11 after an ticipated ovulation tended to increase pregnancy and calving rates in lactating Holstein recipients (Block et al., 2003). The management of these cows was similar to those in Experiment 2. In both studies, recipients were exposed to heat stress and received an IVP embryo using a timed embryo transfer protocol. Effectiveness of treatment with GnRH or its analogues at 11 to12 d after estrus for inseminated cows has yielded variable results, as some reports

PAGE 76

64 indicated a positive effect (S heldon and Dobson, 1993; Willard et al., 2003) while others indicated no effect (Ryan et al., 1994). One f actor that could influence the effectiveness of GnRH treatment at Day 11 is the number of follicular waves that a female experiences during an estrous cycle. Females with estr ous cycles characterized by three follicular waves have larger second-wave dominant follic les at Day 11 than females with two-wave cycles (Ginther et al., 1989; Sa vio et al., 1990; Ko et al., 1991) Given that a follicle must reach 10 mm in diameter to ovulate in re sponse to LH (Sartori et al., 2001), the preponderance of cycle type (two-wave vs three-wave) within a herd may determine effectiveness of GnRH treatment at Day 11. Finally, it remains possible that failure to observe an effect of GnRH treatment was because the number of animals per group was low. The pitfalls associated with interp retation of experiments with low numbers has been discussed (Amann, 2005) and could be resp onsible for the variation in results for trials to test effects of GnRH on pregnancy rates in embryo transfer recipients. Estrus is difficult to detect in lactating dairy cows because of the short duration of estrus and the large proporti on of cows that do not display intense mounting activity (Dransfield et al., 1998). This problem, whic h is exacerbated by heat stress (Thatcher et al., 1986), makes embryo transfer in lactating co ws inefficient if recipient selection is based solely on estrus detect ion. The first report of a tim ed embryo transfer protocol, where ovulation was synchronized using an OvSynch protocol, was by Ambrose et al. (1999). The suitability of timed embryo transf er as a method for prep aring recipients was demonstrated in Experiment 1 because calving rates were 50 and 41% for cow and heifer recipients that received a si ngle embryo, respectively. Similarl y, using beef recipients, a pregnancy rate of 49% was achieved using tim ed embryo transfer (Bo et al., 2002). In

PAGE 77

65 contrast, pregnancy rate at Day 45 of ge station in Experiment 2 was only 17%. Low pregnancy rates have been reported in othe r studies with timed embryo transfer using lactating, heat-stressed recipients with pregnancy rates at ~ 45 d of gestation following timed embryo transfer ranging from 11 – 26% (Ambrose et al., 1999; Al-Katanani et al., 2002a; Block et al., 2003). The reason for th e differences in pregnancy rates between Experiment 1 and 2 cannot be deduced because of the large number of variables between studies including nutrition, housing, level of milk yield, stage of lactation, breed, synchronization protocol, and embryo culture protocol. Despite the effectiveness of timed em bryo transfer, there was a tendency for pregnancy rates in Experiment 1 to be higher fo r those recipients detect ed in estrus. Most of the animals not detected in estrus likely ovulated after the last GnRH injection because embryos were only transferred to recipients with a detectable CL. Nonetheless, some cows in this group probably were not synchr onized with respect to predicted ovulation time. Transfer of IVP embryos has been associ ated with large calf syndrome, increased rates of fetal loss, sex ratio skewed towards the male and increased rate of dystocia and calf mortality (see Hasler et al., 2000; Ha nsen and Block, 2004; Farin et al., 2004 for review). There are also reports of prolonge d gestation length (Kruip and den Dass, 1997; Rerat et al., 2005). In Experiment 1, most char acteristics of the fetus and calf that were measured in females receiving one embryo were within normal ranges including gestation length, rates of fetal loss, calf birth weight, and calf survival at birth and within the first 7 d of age. The incidence of dystocia among fema les receiving one calf was 21% and it is difficult to determine whether this value is high because of the particular mating

PAGE 78

66 combinations used (embryos of diverse ge notypes transferred into females of several different genotypes). In a study with Holsteins bred by artificial insemination, the frequency of difficult births ranged fro m 6 to 18% (Djemali et al., 1987). The one abnormality identified was a skewed sex ratio with 68% of the calves being male. While previous work suggests th at the altered sex ra tio among IVP embryos is due to toxic effects of concentrations of glucose in excess of 1 mM on female embryos (Kimura et al., 2005), the concentration of gluc ose in the medium used for culture here (KSOM-BE2) contains only 0.2 mM glucose (Soto et al., 2 003). Others have found a tendency for male embryos to become blastocysts sooner in development when cultured in KSOM than female embryos (Nedambale et al., 2004b). Differences in sex ratio have been seen as early as between the eight-cell and morula stages of development (Block et al., 2003). While it is possible that selection of most embr yos for transport done on Day 6 after insemination exacerbated the skewed se x ratio, Block et al. (2003) reported that 64% of calves born as a result of transfer of IVP embryos cultured in modified KSOM were male even though embryos were harveste d for transfer on Day 8 after insemination. In conclusion, results indicate that unilate ral transfer of two embryos to increase pregnancy rate is unwarranted. The fact that fetal loss occurred sooner for heifers than cows points out the importance of uterine capacity as a lim iting factor for maintenance of fetal development of two conceptuses. Ther e was also no evidence that GnRH treatment at Day 11 after anticipated ovulation improves pr egnancy rate. Finall y, the suitability of timed embryo transfer as a method for prepari ng recipients for transfer was evident by the high pregnancy and calving rates achieved with crossbred females that received a single embryo. Additional research is warranted to reduce incidence of skewed sex ratio.

PAGE 79

67 While sexed semen could be used to contro l sex ratio (Wilson et al., 2005), it is likely that the underlying biological cau ses of altered sex ratio aff ect other aspects of embryo physiology also.

PAGE 80

68Table 3-1. Effect of recipient type a nd number of embryos transferred per re cipient on pregnancy rates and losses. Recipient type Pregnancy rate, d 64 of gestationab Pregnancy rate, d 127 of gestationac Pregnancy loss between Day 64 and 127 of gestationd Calving ratee,f Pregnancy loss between Day 127 and calvingg Lactating cow – single embryo 8/14 (57%) 8/14 (57%) 0/8 (0%) 7/14 (50%) 1/8 (13%)h Lactating cow – two embryos 6/12 (50%) 2/12 (17%) 4/6 (66%) 2/12 (17%) 0/2 (0%) Nulliparous heifer – single embryo 7/17 (41%) 7/17 (41%) 0/7 (0%) 7/17 (41%) 0/7 (0%) Nulliparous heifer – two embryos 3/15 (20%) 3/15 (20%) 0/3 (0%) 3/15 (20%) 0/3 (0%) a Data are the proportion of animals pregna nt of those that received embryos and, in parentheses, the percent pregnant. b Logistic regression indicated e ffect of recipient type (P=0.07). The odds ratio estimate wa s 0.38 (heifer/cow) (95% Wald CI = 0.13, 1.14; Wald Chi-Square statistic = 2.96, P=0.08). c Logistic regression indicated an effect of number of embryos tran sferred (P<0.03). The odds ratio estimate was 4.13 (one embryo/two embryos) with a 95% Wald CI of 1.243, 13.690. Wald Chi-Square statistic = 5.36; P<0.03). d Data are the proportion of pre gnant recipients at Day 64 that lost their pregnancy by Day 127 of gestation and, in parentheses the percent pregnancy loss. e Data are the proportion of animals that calved of those th at received embryos and, in pare ntheses, the percent pregnant. f Logistic regression indicated an effect of number of embryos tran sferred (P<0.03). The odds ratio estimate was 3.62 (one embryo/two embryos) with a 95% Wald CI of 1.090, 12.047. Wald Chi-Square statistic = 4.41; P<0.04). g Data are the proportion of pre gnant recipients at Day 127 that lost their pregnancy before calving and, in parentheses, the pe rcent pregnancy loss. h One cow expelled a stillborn calf at 251 d of gestation.

PAGE 81

69 Table 3-2. Effect of recipien t type and number of embryos transferred per recipient on characteristics of pregnancy and parturition. Recipient type Gestation length, da Twin pregnanciesb Dystociac Retained placentad Lactating cow – single embryo 282 + 3 0/7 (0%) 2/7 (29%) 4/7 (57%) Lactating cow – two embryos 274 + 5 1/2 (50%) 0/2 (0%) 1/2 (50%) Nulliparous heifer – single embryo 276 + 3 0/7 (0%) 1/7 (14%) 5/7 (71%) Nulliparous heifer – two embryos 284 + 4 1/3 (33%) 1/3 (33%) 2/3 (67%) a Data are least-squares means + SEM. Gestation length was a ffected by recipient type x number of embryos transferred (P<0.05). b Data are the proportion of pregnancies in which twin calves were born and, in parentheses, the percent pre gnant. Logistic regression indicate d an effect of number of embryos transferred (P<0.02). c Data are the proportion of pregnancies in wh ich dystocia was recorded at birth and, in parentheses, the percent cows experiencing dystocia. d Data are the proportion of cows calving that experienced retained placenta and, in parentheses, the percent cows experiencing retained placenta.

PAGE 82

70 Table 3-3. Effect of recipien t type and number of embryos transferred per recipient on characteristics of calves born. Recipient type Sex ratio (M:F)a Calf birth weight, kgb Calf mortality at birthc Calf mortality to Day 7 of aged Lactating cow – single embryo 5:3e 34 + 3 0/7 (0%) 0/7 (0%) Lactating cow – two embryos 2:1 25 + 5 0/3 (0%) 0/3 (0%) Nulliparous heifer – single embryo 4:3 26 + 3 1/7 (14%)f 0/6 (0%) Nulliparous heifer – two embryos 4:0 25 + 5 3/4 (75%)g 0/1 (0%) a The overall sex ratio of 15 male and 7 female s tended to be different (P<0.10) than the expected 1:1 ratio. b Data are least-squares means + SEM. c Data are the proportion of calves that were born dead and, in parentheses, the percent born dead. d Data are the proportion of calves born alive that died before d 7 of live and, in parentheses, the percent death before Day 7. e Data includes the stillborn calf at 251 d of gestation f One calf was stillborn from a cow not experiencing dystocia. g One heifer had twin fetuses and both were bor n dead as a result of complications with calving. The other two heifers ga ve birth to a single calf. On e calf was born alive and the other was born dead as a result of complications with calving.

PAGE 83

71 Air temperature ( o C) 10 15 20 25 30 35 Nov 1Nov 15Dec 15 Dec 1 Dec 30Experiment 1 BoliviaRH (%) X Data 0102030405060708090100110120130140150160 60 70 80 90 100 Air temperature ( o C) 5 10 15 20 25 30 35 June 1July 1Aug 1Sept 1Oct 1Nov 1Experiment 2 Florida Figure 3-1. Maximum (open circ les) and minimum (closed circ les) daily air temperatures and relative humidities (RH) during the experiments.

PAGE 84

72 CHAPTER 4 EFFECTS OF HYALURONIC ACID IN CULTURE AND CYTOCHALASIN B TREATMENT BEFORE FREEZING ON SURVIVAL OF CRYOPRESERVED BOVINE EMBRYOS PRODUCED IN VITRO Introduction In vitro production of embryos is an impor tant tool for improving genetic merit and fertility of cattle and is an indispensable co mponent of other technologies such as somatic cell cloning and transgenesis (Hansen and Block, 2004). One limitation to the widespread use of in vitro produced embr yos in the cattle industry is the poor survivability of in vitro produced embryos to cryopreservation. In vitro survival rates following thawing (Pollard and Leibo, 1993; Enright et al., 2000; Khurana and Niemann, 2000a; Diez et al., 2001; Guyader-Joly et al ., 1999) and pregnancy rates following thawing and transfer (Hasler et al., 1995; Agca et al., 1998; Ambrose et al., 1999; AlKatanani et al., 2002a) are c onsistently lower for embryos produced in vitro when compared to embryos produced in vivo by superovulation. The poor survival of the in vitro produced embryo is associated with cultureinduced changes in ultrastructure (Rizos et al., 2002), gene expressi on (Bertolini et al., 2002a; Lazzari et al., 2002; Lonergan et al., 2003), and metabolism (Krisher et al., 1999; Khurana and Niemann, 2000b) that make it dist inct from the embryo produced in vivo. Among the metabolic changes are an increase in lipid content (Abe et al., 1999; Rizos et al., 2002) and this condition has been linked to poor freezability. Mechanical delipidation (Tominaga et al., 2000; Diez et al., 2001) and ad dition of inhibitors of fatty acid synthesis (De la Torre-Sanchez et al., 2005) can im prove survival following cryopreservation.

PAGE 85

73 In the current study, two approaches for enhancing survival of bovine embryos following cryopreservation were evaluated. Th e first was to culture embryos in the presence of hyaluronic acid. This unsulphate d glycosaminoglycan is present in follicular, oviductal and uterine fluids in several species including catt le (Lee and Ax, 1984). Receptors for hyaluronic acid (CD44) have been reported on the bovine oocyte, cumulus cell, and preimplantation stage embryo (Valca rcel et al., 1999). Addition of hyaluronic acid to culture medium has been reported to increase blastocy st re-expansion and hatching after freezing (Stoj kovic et al., 2002; Lane et al ., 2003). The second approach was to determine whether altering the cy toskeleton before cryopreservation would enhance embryo survival. The rationale for this treatment is that cryoinjuries such as intracellular ice formation and osmotic s hock induce irreversible disruption in microtubules and microfilaments (Kuwayama et al., 1994; Fair et al., 2001) and that temporary depolymerization of actin micr ofilaments before cryopreservation could reduce cytoskeletal damage and plasma me mbrane fracture caused by alterations in cytoskeletal architecture (Dobrinsky, 1996). A ddition of cytochalasin B to cause actin depolymerization had no effect on survival of eight-cell embryos in the mouse (Prather and First, 1986) but enhanced survival of expanded and hatched blastocysts without effecting survival of morula and early blas tocysts in the pig (Dobrinsky et al., 2000). Materials and Methods Embryo Production Procedures, reagents, and media formulation for oocyte maturation, fertilization, and embryo culture were as previously de scribed (Roth and Hansen, 2005) with some modifications. Briefly, cumulus oocyte comple xes (COCs) were harvested from ovaries of a variety of breeds collected at a local abattoir located at a travel distance of

PAGE 86

74 approximately 1.5 h from the laboratory. The COCs were matured in Tissue Culture Medium-199 with Earle’s salts supplemented with 10% (v/v) steer serum, 2 g/mL estradiol 17, 20 g/ml follicle stimulating horm one, 22 g/ml sodium pyruvate, 50 g/ml gentamicin and an additional 1 mM gl utamine for approximately 22 h at 38.5C in an atmosphere of 5% (v/v) CO2 in humidified air. Inse mination with a cocktail of Percoll-purified spermatozoa from three different bulls was performed in In Vitro Fertilization – Tyrode’s Albumin Lactat e solution. At 8 – 12 h post-insemination (hpi), putative zygotes were denuded of cumulus cells by suspension in Hepes-TALP medium containing 1000 units/ml hyaluronidase type IV (Sigma, St Louis, MO, USA) and vortexing in a microcentrifuge tube for 5 min. Presumptive zygotes were then placed in groups of ~30 in 50 l microdrops of a modified S ynthetic Oviductal Fluid (SOF) prepared as described by Fisher-Brown et al (2002). Embryos were cultured at 38.5C in a humidified atmosphere of 5% (v/v) CO2, 5% O2, and with the balance N2. Blastocysts were collected for cryopreservat ion on day 7 after insemination. Experimental Design and Embryo Manipulation The experiment was a 2 x 2 factorial design to test main effects of hyaluronic acid during culture (+ or -) and cytochalasin B before cryopres ervation (+ or -). Data on development were obtained from 18 repl icates using 5022 oocytes while data on cryopreservation were obtained from 7 rep licates using a total of 197 blastocysts. Following insemination and transfer to fr esh microdrops, embryos cultured without hyaluronic acid were cultured in SOF for 7 days beginning after insemination. Embryos treated with hyaluronic acid were cultured in SOF until day 5 when all embryos were transferred to a fresh microdrop of SOF containing 6 mg/ml hyaluronic acid from Streptococcus zooepidemicus (Sigma).

PAGE 87

75 Blastocysts and expanded blastocysts were harvested on the morning of day 7 after insemination and washed twice in holding medi um consisting of Hepes-TALP (Parrish et al., 1989) containing 10% (v/v) fetal calf serum (FCS). Embryos treated with cytochalasin B were incubated for 10 mi n at 38.5oC in air while in Hepes-TALP containing 10% (v/v) FBS and 7.5 g/ml cytochalasin B (Sigma) in a 1.5 ml microcentrifuge tube (Tominaga et al., 2000). Cytochalasin B was initially dissolved in DMSO at a concentration of 5 mg/ml and wa s then added to HEPES-TALP to achieve a final concentration of 7.5 g/m l. Control embryos were incubated similarly in HEPESTALP containing 10% (v/v) FBS. Cryopreservation Procedures for freezing were modified from those reported elsewhere (Hasler et al., 1995; Enright et al., 2000). In brief, blastocysts were transferred in groups of 10 to a fresh 100 l microdrop of Hepes-TALP containing 10% FCS at 38.5oC for the time it took to harvest all embryos (~ 10 min). Next, em bryos in groups of 5 8 per treatment (hyaluronic acid or control) we re randomly selected to receive cytochalasin B treatment before freezing or not as described above. Af terwards, each group of 5 – 8 embryos was placed in a 50 l microdrop of 10% (v/v) glycerol in Dulbecco’s phosphate-buffered saline (DPBS) containing 0.4% (w/v) bovine serum albumin (freezing medium) in a grid plate over a slide warmer at 30oC. Within 10 min, embryos were loaded in a 50 l volume into 0.25 ml plastic straws (Agtech, Manhattan, KS). Up to 8 embryos were loaded in each straw. Two columns of 50 l freezing medi um separated by air bubbles were always placed above and below the column of embryos Straws were transferred to a freezing chamber (Cryologic Model CL5500 (Mulgrave, Victoria, Australia) for 2 min at -5oC and then ice crystals were induced by touching the straw where the top column of medium

PAGE 88

76 resided with a cotton plug that had been immersed in liquid nitrogen. After an additional 3 min at -5oC, embryos were cooled to -32oC at a rate of -0.6oC/min. After 2 min at 32oC, straws were directly immersed in liquid N2 and stored until thawing (4 days – 1 week later). Thawing and Determination of Survival Straws containing embryos were thawed by warming for 10 sec in air at room temperature and 20 sec in a 32oC water bath. All subsequent steps before culture were performed with media prewarmed to ~30oC and with dishes placed on a slide warmer set at 30oC. Embryos were then expelled into an empty petri dish and immediately transferred to a fresh 60 l drop of DPBS containing 6.6% (v/v) glycerol and 0.3 M sucrose in an grid dish. After 5 min, embr yos were sequentially transferred to DPBS containing 3.3% (v/v) glycer ol and 0.3 M sucrose for 5 min and DPBS + 0.3 M sucrose for 5 min. Embryos were then washed thre e times in HEPES-TALP + 10 % (v/v) FCS and placed into culture in groups of 58 in 25 l microdrops of SOF containing 10% (v/v) FCS. Culture was at 38.5C in a humidified atmosphere of 5% (v/v) CO2, 5% O2, and 90% N2. Re-expansion was determined at 48 h after thawing and hatching at 72 h. Statistical Analysis The proportion of oocytes that cleaved and the proportion of embryos that developed to the blastocyst stage on day 7 and day 8 were determined for each replicate. Treatment effects were determined by least-sq uares analysis of vari ance using the proc GLM procedure of SAS (SAS for Windows 90, Ca ry, NC). The model included the main effects of replicate and treatment. Data fo r the proportion of frozen/thawed embryos that re-expanded and on the proportion that hatche d by 72 h of culture were analyzed using the CATMOD procedure of SAS. The initia l model included all main effects and two-

PAGE 89

77 way interactions. After removing nonsigni ficant effects, the final model included replicate, hyaluronic acid, preparation prior to freezing (none, cytochalasin B), and the interaction of hyaluronic acid a nd preparation before freezing. Results Effect of Hyaluronic Acid on Embryonic Development As shown in Table 1, addition of hyaluroni c acid at day 5 after insemination caused a slight reduction in the yi eld of blastocysts on day 7 and day 8 after insemination regardless of whether data were expressed as the proportion of oocyt es developing to the blastocyst stage (P < 0.05) or the pro portion of cleaved embryos developing to the blastocyst stage (P < 0.01). Of the blas tocysts that were recovered, 62-68% were recovered at day 7 and the balance at day 8. There was no effect of hyaluronic acid on the proportion of blasto cysts collected at da y 7 (Table 4-1). Survival after Cryopreservation Overall, cytochalasin B increased th e percent of embryos that re-expanded following thawing (P < 0.0001) a nd that hatched following th awing (P < 0.05) (Table 42). Re-expansion rates were 51.2% (22/43) for embryos treate d with cytochalasin B and 18.2% (8/44) for embryos not subjected to cy tochalasin B. Hatc hing rates were 39.5% (17/43) for embryos treated with cytochal asin B and 4.5% (2/44) for embryos not subjected to cytochalasin B. While there was no significant effect of hyaluronic acid on cryosurvival, there was a tendency (P=0.09) for a hyaluronic acid x cyto chalasin B interaction affecting percent of blastocysts that hatched following thawing. This interaction re flects the fact that hyaluronic acid increased the percent hatching for embryos not subjected to cytochalasin B treatment and decreased percent hatched fo r embryos subjected to cytochalasin B.

PAGE 90

78 Discussion Of the two treatments evaluated for enhanc ing cryosurvival of in vitro produced bovine embryos, cytochalasin B treatment was the most effective as determined by an improvement in both embryo re-expansion and ha tching. The rationale for this treatment is to reduce cellular injury caused by di sruption in microtubules and microfilaments (Kuwayama et al., 1994; Fair et al., 2001) a nd to increase flexibility of the plasma membrane to allow it to tolerate forces a ssociated with freezing that lead to membrane damage. In other studies, addition of cytochal asin B had no effect on survival of eightcell embryos in the mouse (Pra ther and First, 1986), enhan ced survival of expanded and hatched pig blastocysts without effecting survival of mo rula and early blastocysts (Dobrinsky et al., 2000), and improved surviv al of in vivo derive d bovine blastocysts subjected to vitrification (Dobrinsky et al., 1995). For embryos not exposed to cytochalasin B, there was a tendenc y for those cultured in hyaluronic acid to have a higher re-expansion rate and hatching rate than embryos cultured without hyaluro nic acid. Both Stojkovic et al (2002) and Lane et al. (2003) reported improved survival rates to freezing when embryos were cultured in hyaluronic acid; such a beneficial e ffect has not always been observed (Furnus et al., 1998). Surprisingly, embryos cultured in hyaluronic acid were less likely to survive freezing than control embryos when the cytochalasin B treatment was applied. Perhaps physiological changes induced by hyaluronic ac id cause the embryo to be less able to adjust to the cellular actions of cytochalas in B. Those changes are potentially numerous because hyaluronic acid acts to affect cell function through several means including signaling through cell surface receptors, m odifying the biophysical properties of extracellular and pericellular matrices by at tracting water, and by interacting physically

PAGE 91

79 with a variety of ions and other molecu les (Laurent, 1987; Ruoslahti and Yamaguchi, 1991; Hardingham and Fosang, 1992; Yasuda et al., 2002; Toole et al., 2005). One possible mechanism by which hyaluronic acid could increase embryo survival to freezing is by increasing the total number of cells in the embryo (Sto jkovic et al., 2002; Jang et al., 2003; Kim et al., 2005) One unexpected finding was the reduction in the percentage of embryos that became blastocysts caused by hyaluronic acid. In other studies, hyaluronic acid either had no effect (Stojkovic et al., 2002; Lane et al., 2003) or caused an increase in blastocyst yield (Furnus et al., 1998; Jang et al., 2003). Differences in origin and concentration of hyaluronic acid could explain some of this difference between studi es. Hyaluronic acid can be isolated from different sources (ex., bacteria, rooster comb, and umbilical cord) and preparations can differ in protein, endotoxin, and nucleotide content (Shiedlin et al., 2004). Stojkovic et al. (2002) reported that preliminary re sults indicated that embryo development in vitro was dependent upon th e origin of the commercially-available hyaluronic acid. However, embryos cultured w ith hyaluronic acid experienced a change in culture medium at day 5 whereas contro l embryos did not. Such a difference could have obscured beneficial effects of hyaluro nic acid although anothe r paper indicates no effect of changing culture medium at 72 hpi on blastocyst yield in cattle (Ikeda et al., 2000). The percent of embryos that underwent ha tching after freezing in glycerol and thawing has varied from 0% (Enright et al., 2000) 22% (Diez et al., 2001; Nedambale et al., 2004a), 32% (Guyader-Joly et al., 1999) and 69% (Hasle r et al., 1997). The best survival achieved in this study was for em bryos cultured without hyaluronic acid and

PAGE 92

80 treated with cytochalasin B. In this group, 51.2% of cryopreserved embryos were capable of re-expansion and 39.5% hatched. It is likely that the percent hatching can be further improved by modifying post-thaw cult ure-conditions. Massi p et al. (1993) found hatching rates for frozen/thawed, in vitro pr oduced embryos were 41% when culture was performed in the presence of bovine oviductal epithe lial cells while ha tching rate using other culture conditions not i nvolving co-culture was 0-6%. Nonetheless, one would not expect optimal pregnancy rates to be achie ved following direct transfer of embryos frozen in glycerol even with the inclusion of cytochalasin B treatment. Rather, it is suggested that pregnancy rate s following transfer of embr yos cryopreserved using slowfreezing procedures can be optimized by se lecting embryos for transfer based on development in culture shortly after thawing. In contrast to the poor survival of in vitro-produced embryos frozen using conventional slow-freezing techniques, severa l experiments indicate that cryosurvival can be enhanced by using vitrification (Vajta, 2000). It remains to be tested whether survival of embryos produced in vitro after vitrif ication can be improve d by cytochalasin B treatment. There was a beneficial effect of cytochalasin B treatment on cryosurvival of embryos derived in vivo following vitrif ication (Dobrinsky et al., 1995). In conclusion, cytochalasin B treatment before freezing improved cryosurvival of bovine embryos produced in vitro and subjecte d to slow-freezing in glycerol. Such a treatment could be incorpor ated into methods for cryopr eservation of bovine embryos provided post-transfer survival is adequate. In contrast, culture with hyaluronic acid was of minimal benefit the increa sed cryosurvival in the absence of cytochalasin B was not sufficient to allow an adequate num ber of embryos to survive.

PAGE 93

81 Table 4-1. Effect of hyaluronic acid added at day 5 after insemination on production of blastocysts at day 7 a nd 8 after inseminationa,b. Culture medium Number of oocytes Percent cleaved Blastocysts/oocyte (%) c Blastocysts/cleaved embryo (%) c Percent of total blastocysts that were collected at day 7 Control 1935 76.0 + 0.9 36.0 + 1.2* 47.2 + 1.3** 68.8 + 2.4 Hyaluronic acid 3087 77.7 + 0.9 31.5 + 1.2 40.7 + 1.3 62.2 + 2.4 a n=18 replicates b Means within a column that differ significantly are indicated by (P < 0.05) and ** (P < 0.01) c Includes blastocysts collected at da y 7 and those collected at day 8. Table 4-2. Effect of culture in hyaluronic acid and treatment with cytochalasin B on survival after cryopreservation. a Culture medium Cytochalasin treatment Re-expansion by 72 hb Hatching by 72 hc Control Control 8/44 (18.2%) 2/44 (4.5%) Control Cytochalasin B 22/43 (51.2%) 17/43 (39.5%) Hyaluronic acid Control 16/55 (29.0%) 7/55 (12.7%) Hyaluronic acid Cytochalasin B 26/55 (47.3%) 12/55 (21.8%) a Data are the fraction of embryos, and in parentheses, percent. Number of replicates was 7. b Effect of cytochalasin B (P < .0001). c Effect of cytochalasin B (P < 0.05), hyaluronic acid (P < 0.10), and the cytochalasin B x hyaluronic acid interaction (P = 0.09).

PAGE 94

82 CHAPTER 5 GENERAL DISCUSSION As alluded to at the beginning of this thes is, there has been a pr ecipitous decline in fertility of dairy cows over the last 1040 years in North America (Butler, 1998), Ireland (Roche, 2000), Spain (Lpez-Gatius et al., 2003 ), and the United Kingdom (Royal et al., 2000). In addition, heat stress can compromise fe rtility in lactating dairy cows (Putney et al., 1989b; Al-Katanani et al., 1999). The purpos e of the present series of experiments described in the thesis was to 1) evaluate st rategies for enhancing fe rtility after AI using GnRH treatment (Chapter 2) and 2) further develop ET usin g in vitro produced embryos as a tool for increasing fertility by testing whether pregnancy rate could be improved by transfer of twin embryos (Chapter 3) a nd whether the developmental competence of embryos after cryopreservation could be improved by hyaluronan or cytochalasin B treatment (Chapter 4). Results indicated no c onsistent benefit of in jection of GnRH at Day 11-15 after anticipated ovulation or inse mination on pregnancy rates in heifers or lactating cows. While unilateral transfer of tw o embryos was not shown to be an effective treatment for increasing pregna ncy rate in recipients, the high pregnancy rates achieved in this study point to the pot ential usefulness of ET as a tool for enhancing fertility. Large-scale use of embryo transfer will requi re the ability to freeze embryos successfully. Results suggest that treatment of embryos with cytochalasin B before freezing is a promising tool for enhancing survival of embryos following cryopreservation. A large number of studies have been performed to te st the effect of GnRH administration after expected ovulation on fertility of cattle. Previous results indi cated that GnRH was

PAGE 95

83 sometimes effective at increasi ng pregnancy rate, but this bene ficial effect was often not observed (Peters et al., 2000). Despite th is knowledge, we chose to reevaluate the effectiveness of GnRH treatment because of a report that GnRH treatme nt at Day 11 after estrus increases pregnancy rate s in lactating cows exposed to heat stress (Willard et al., 2003). Accordingly, it was hypothesized in Chapter 2 that the beneficial effect of GnRH treatment would be greater during the summer than winter. This may be so because the antiluteolytic process may be compromised by heat stress because of decreased growth of the filamentous stage conceptus (Biggers et al., 1987) and increased uterine PGF2 secretion from the uterus (Wolfenson et al., 1993). Overall, the results of GnRH treatment we re generally negative. For treatment at Day 11, a positive effect of GnRH on fertil ity was never seen. This was the case for heifers and lactating cows subjected to AI or whether animals were exposed to heat stress or not (Chapter 2; experiment 1 and 2). Treatment of lacta ting recipients with GnRH at Day 11 also failed to increase pregnancy rate dur ing heat stress in ET recipients (Chapter 3, experiment 2). Effectiveness of treatmen t with GnRH or its analogues at 11 to 12 d after estrus for inseminated, heat-stressed lact ating cows has yielded variable results, as some reports indicated a positive effect (She ldon et al., 1993; Willard et al., 2003), while others indicated no effect (Jubb et al., 1990). Also, admini stration of GnRH at Day 11 after anticipated ovulation tended to increase pregnancy and calving rates in lactating Holstein embryo transfer reci pients exposed to heat stre ss (Block et al., 2003). One factor that could influence the effec tiveness of GnRH treatment at Day 11 is the number of follicular waves that a female experiences during an estrous cycle. Females with estrous cycles characterized by three follicular waves have larger second-wave

PAGE 96

84 dominant follicles at Day 11 than females w ith two-wave cycles (Ginther et al., 1989; Savio et al., 1990; Ko et al., 1991) Given that a follicle must reach 10 mm in diameter to ovulate in response to LH (Sartori et al., 2001), the preponderance of cycle type (twowave vs three-wave) within a herd may dete rmine effectiveness of GnRH treatment at Day 11. In one experiment (Chapter 2; experime nt 3), administration of GnRH at Day 14 after anticipated ovulation in cows subject ed to TAI increased pregnancy rates of lactating cows in the summer and winter at two locations. In the following year, though, GnRH failed to improve fertility when treatm ent was administered either at day 14 in cows subjected to TAI (experiment 4) or at day 14 or 15 in cows previously diagnosed coming in estrus (experiment 5). It is impor tant to recognize that GnRH treatment should improve fertility only when triggering luteinization or ovulation of developing (estrogenic) follicles. Thus, there are at leas t two possible reasons for a lack in response upon GnRH treatment at day 14 or 15. One possi bility relates to the timing of ovulation relative to the GnRH treatment and whether these animals failed to ovulate after being diagnosed as coming into estrus. Although af ter observing estrus one does not expect ovulation to fail, this expression does not necessarily mean that subsequent ovulation occurred (Lpez-Gatius et al., 2005b) and inse mination after a false identified estrus often occurs (Heersche and Nebel 1994). According to Lpez-Gatius et al. (2005b), the risk of cows failing to ovulate (12%) duri ng the summer was greater than in the cool period (3%). During experiment 3 all cows received a GnRH injection at 72 h following PGF2 to insure an ovulation of the synchronized dominant follicle. Perhaps, the positive

PAGE 97

85 GnRH effect observed during experiment 3 was masked in the following experiment because cows did not receive an additional GnRH dose at estrus to ensure subsequent ovulation. According to Lopez-Gatius et al. ( 2005a), there is evid ence demonstrating the benefits upon GnRH treatment when given on the day of insemination compared to controls (30.8% vs. 20.6%), but conception rates were grea ter if cows received an additional dose at day 12 post-inseminati on (35.4%). On the other hand, when GnRH treatment took place on day 15 to ensure a re sponsive (estrogenic) dominant follicle would ovulate at the time of GnRH treatment it failed to improve fertility as well. Similarly, in a recent study (Bartolome et al., 2005) there was no effect of GnRH treatment on pregnancy rates of lactating cows when administered either on day 15 or day 5 and 15 after TAI. It remains possible that inconsistency in effects of GnRH treatment is caused in part by the low number of animals per treat ment group. The pitfalls associated with interpretation of experiments with low numb ers has been discussed (Dransfield et al., 1998) and could be responsible for the variation in results for trials to test effects of GnRH on pregnancy rates in embryo transfer recipients and for inseminated cows. With an existent variation among trials regarding the use of GnRH at day 11-15 post-insemination, one could specu late that such inconsistenc y regarding treatment is due to the fact that herds of cattle determine th e result that an experiment achieves. However, our results indicate that such a hypothesis is not likely becau se when an experiment was replicated the next year using the same he rd, GnRH treatment once again proved to be inconsistent in improving pregnancy rates.

PAGE 98

86 According to Thatcher et al. (2005), hCG results in a more prolonged rise in LH activity than is achieved fo llowing GnRH treatment. Perhap s the likelihood of ovulating or luteinizing the dominant follicles presen t at the time of treatment would be higher using hCG. Although low numbers of insemina ted animals were used (n=8; n=49) hCG treatment on d 14 after estrus improved pregnancy rates (Rajamahendran and Sianangama, 1992; Sianangama and Rajamahe ndran, 1992). Use of hCG warrants further investigation for any additional effect or response during the summer to enhance pregnancy rates of lactating cows. Recent work has focused on use of ET to bypass early embryonic death (Putney et al., 1989b; Ambrose et al., 1999; Al-Katanani et al., 2002a). Given that ET can be more effective at increasing pregnanc y rates than AI for lactating cows during periods of heat stress (Putney et al., 1989b; Ambrose et al., 1999; Drost et al., 1999; Al-Katanani et al., 2002a), the potential benefit of ET can be realized. For ET to become an economical alternative to AI on a wide scale basis in co mmercial herds, embryos must be inexpensive to produce (Hansen and Block et al., 2004) Although embryos produced using IVP systems are relatively inexpe nsive as compared to embr yos produced by superovulation, pregnancy rates achieved following transfer of an IVP embryo are often less than what is obtained following transfer of an embryo produced by superovulation (Hasler et al., 1995; Agca et al., 1998; Ambrose et al., 1999; Al-Katanani et al., 2002a). In addition, IVP embryos are less likely to survive freezing than superovulated embryos (Hasler et al., 2003), likely due to their increa sed lipid content (Abe et al ., 1999; Rizos et al., 2002). Accordingly, the second approach for the thesis focused on improvements in ET by

PAGE 99

87 comparing pregnancy rates following the tran sfer of two embryos compared to one and by increasing the viability of embryos that were cryopreserved. The first effort was to determine whether transfer of two IVP embryos into the uterine horn ipsilateral to the CL could increase pregnancy rates during periods of heat stress. It was hypothesized that such a treatment might incr ease pregnancy rates because the likelihood is increased that the cow receives at least one embryo competent for sustained development. In addition, the tran sfer of two embryos into the ipsilateral uterine horn is likely to incr ease the amounts of interferonand other embryonic signaling molecules in the uterus needed to ma intain pregnancy and prevent luteolysis. Transferring two embryos in to the uterine horn ipsilateral to the CL failed to increase pregnancy rates. Instead, the transf er of two embryos into recipients led to pregnancy loss, which occurred earlier fo r heifers than for cows. The most likely explanation for the increased frequency of pregnancy loss in recipients receiving two embryos is uterine crowding, with the effect s of crowding occurring sooner in gestation for nulliparous animals than for multiparous animals. Regardless of whether twin transfers were performed via bilateral or unilateral placement, similar results were obtained in another study (Ande rson et al., 1979). In contra st, calving rate and twinning rate in heifers was lower for unilateral twin transfers than for bilateral transfers. Similarly, Rowson et al. (1971) also found lo wer embryonic survival rates and twinning rates for recipients of unilateral twin transfers than for recipients of bilateral transfers in heifers. It is evident that uterine cap acity can vary between herds of cattle. Thus, there were no differences in pregnancy success between recipients of twin embryos placed

PAGE 100

88 unilaterally or bilaterally for heifers (Sr eenan et al., 1989, Reiche nbach et al., 1992) or cows (Sreenan et al., 1989). Similarly, embryoni c survival rate for beef cows selected for twinning was similar for those having un ilateral or bilatera l multiple ovulations (Echternkamp et al., 1990). In lactating dairy cows, in cont rast, the likelihood of a twin pregnancy resulting from multiple ovulations going to term was higher if ovulations occurred bilaterally than if unilateral ovul ations occurred (Lpez-Gatius and Hunter, 2005). Perhaps, identification of the biologi cal processes controlling uterine capacity will lead to new approaches for increasing th e efficacy of producing twins in cattle. An additional limitation to the widespread use of IVP embryos in cattle is their poor survival following cryopreservation. In vitro survival rates following thawing (Pollard and Leibo, 1993; Enright et al., 2000; Khurana and Niemann, 2000a; Diez et al., 2001; Guyader-Joly et al., 1999) and pregnancy rates follo wing thawing and transfer (Hasler et al., 1995; Agca et al., 1998; Ambr ose et al., 1999; Al-Kat anani et al., 2002a) are consistently lower for IVP embryos when compared to embryos produced in vivo by superovulation. The percent of embryos that underwent ha tching after freezing in glycerol and thawing has varied from 0% (Enright et al ., 2000), 22% (Diez et al., 2001; Nedambale et al., 2004a), 32% (Guyader-Joly et al., 1999), and 69% (Hasler et al., 1997). Of the two treatments evaluated for enhancing cryosurvi val of IVP bovine embryos, cytochalasin B treatment was the most effective as dete rmined by an improvement in embryo reexpansion and hatching rates. In this tr eatment, 51.2% of cryopreserved embryos were capable of re-expansion and 39.5% hatched. No netheless, one would not expect optimal

PAGE 101

89 pregnancy rates to be achieved following direct transfer of embryos frozen in glycerol even with the inclusion of cytochalasin B treatment. In contrast to the poor survival of IV P embryos frozen using conventional slowfreezing techniques, several experiments indica ted that embryo survival can be enhanced following vitrification (Vajta, 2000) It remains to be tested whether survival of embryos produced in vitro after vitrific ation can be improved by cytoch alasin B treatment. Rather, it is suggested that pregnancy rates followi ng transfer of embryos cryopreserved using slow-freezing procedures can be optimized by selecting embryos for transfer based on development in culture shortly after thawing. Indeed, fertility issues will continue to dr ive new ideas for developing strategies to improve or at least reduce undesirable con ception and pregnancy rates in any cattle operation. Efficiency among cattle operations is of major interest and ET has the potential to be the vehicle th at can help overcome some fer tility issues associated with oocyte developmental competence, fertiliz ation, and early embryonic development. However, the potential this reproductive technology has is underestimated when pregnancy rates continue to be less than AI during the absence of heat stress. Further research that identifies embryos that are more likely to survive following transfer and establish a pregnancy is warranted. In conclusion, GnRH treatment did not c onsistently increase pregnancy rates when administered at Day 11-15 after insemination and is not recommended as a fertilityenhancing treatment. Similarly, tr ansfer of two embryos to th e uterine horn ipsilateral to the CL was not an effective method for increas ing pregnancy rates in recipients. Transfer of cryopreserved embryos may be enhanced by treatment of embryos with cytochalasin B

PAGE 102

90 since this molecule increased in vitro surviv al. Since several experi ments indicate that cryosurvival can be enhanced using vitrifi cation (Vajta, 2000), it remains to be tested whether survival of IVP embryos after vitr ification can be improved by cytochalasin B treatment.

PAGE 103

91 LIST OF REFERENCES Abe H, Yamashita S, Itoh T, Satoh T, Hoshi H. Ultrastructure of bovine embryos developed from in vitro-matured and fertil ized oocytes: comparative morphological evaluation of embryos cultured either in serum-free medium or in serum supplemented medium. Mo l Reprod Dev 1999; 53:325-35. Abilay TA, Johnson HD, Madan M. Influen ce of environmental heat on peripheral plasma progesterone and cortisol during th e bovine estrous cycle. J Dairy Sci 1975; 58:1836-40. Achard D, Gilbert M, Bnistant C, Slam a SB, DeWitt DL, Smith WL and Lagarde M. Eicosapentaenoic and docosahexaenoic acid s reduce PGH synthase 1 expression in bovine aortic endothelial cells. Bi och Biophy Res Comm 1997; 241:513–18. Adam CL, Findlay PA, Moore HA. Eff ects of insulin-like growth factors-I on luteinizing hormone secretion in sh eep. Anim Reprod Sci 1997; 50:45-56. Agca Y, Monson RL, Northey DL, Abas Mazni O, Schaeffer DM, Rutledge JJ. Transfer of fresh and cryopreserved IVP bovine embryos: normal calving, birth weight and gestation lengths. Theriogenology 1998; 50:147-62. Ahlswede L, Lotthammer KH. Studies on a spec ific vitamin A-unrelated effect of beta carotene on the fertility of ca ttle. 5. Studies of organs (ovaries, corpora lutea, liver, fatty tissues, uterine secretion, adrena l glands--determination of weight and content] Dtsch Tierarztl Wochenschr 1978; 85:7-12. Ahmad N, Townsend EC, Dailey RA, Inskeep EK Relationships of hormonal patterns and fertility to occurrence of two or th ree waves of ovarian follicles, before and after breeding, in beef cows and he ifers. Anim Reprod Sci 1997; 49:13-28. Akordor FY, Stone JB, Walton JS, Leslie KE, Buchanan-Smith JG. Reproductive performance of lactating Hols tein cows fed supplemental -carotene. J Dairy Sci 1986; 69:2173-8. Al-Katanani YM, Webb DW, Hansen PJ. Fact ors affecting seasonal variation in 90 day non-return rate to first service in lactating Holstein cows in a hot climate.J Dairy Sci 1999; 82:2611-5. Al-Katanani YM, Drost M, Monson RL, Rutledge JJ, Krininger-III CE, Block J, Thatcher WW, Hansen PJ. Pregnancy rates following timed embryo transfer with fresh or vitrified in vitro produced embryos in lactating dairy cows under heat stress conditions. Theriogenology 2002a; 58:171-82.

PAGE 104

92 Al-Katanani YM, Paula-Lopes FF, Hansen PJ. Effect of season and exposure to heat stress on oocyte competence in Holste in cows. J Dairy Sci 2002b; 85:390-6. Alvarez RH, da Silva MV, de Carvalho JB, Bi nelli M. Effects of inbreeding on ovarian responses and embryo production from s uperovulated Mantiqueira breed cows. Theriogenology 2005; 64:1669-76. Amann RP. Weaknesses in reports of "f ertility" for horses and other species. Theriogenology 2005; 63:698-715. Ambrose JD, Drost M, Monson RL, Rutledge JJ, Liebfried-Rutledge ML, Thatcher MJ, Kassa T, Binelli M, Hansen PJ, Chenoweth PJ, Thatcher WW. Timed embryo transfer in heat-stressed dairy cattle: A field trial with IVF-derived embryos. J Dairy Sci 1997; 80:239 (Abstr.). Ambrose JD, Drost M, Monson RL, Rutledge JJ, Leibfried-Rutledge ML, Thatcher MJ, Kassa T, Binelli M, Hansen PJ, Chenoweth PJ, Thatcher WW. Efficacy of timed embryo transfer with fresh and frozen in vitro produced embryos to increase pregnancy rates in heat-stressed da iry cattle. J Dairy Sci 1999; 82:2369-76. Anderson GB, Cupps PT, Drost M. Induction of twins in cattle with bilateral and unilateral embryo transfer. J Anim Sci 1979; 49:1037-42. Archiga CF, Vzquez-Flores S, Ortz O, He rnndez-Cern J, Porras A, McDowell LR, Hansen PJ. Effect of injection of -carotene or vitamin E and selenium on fertility of lactating dairy cows. Theriogenology 1998a; 50:65-76. Archiga, CF, Staples CR, McDowell LR, Hansen PJ. Effects of timed insemination and supplemental -carotene on reproduction and milk yield of dairy cows under heat stress. J Dairy Sci 1998b; 81, 390-402. Armstrong JD, Goodall EA, Gordon FJ, Rice DA, McCaughey WJ. The effects of levels of concentrate offered and incl usion of maize gluten or fi sh meal in the concentrate on reproductive performance and blood pa rameter of dairy cows. Anim Prod 1990; 50:1–10. Badinga L, Thatcher WW, Diaz T, Drost M, Wo lfenson D. Effect of environmental heat stress on follicular steroidogenesis and de velopment in lactating Holstein cows. Theriogenology 1993; 39:797-810. Badinga L, Thatcher WW, Wilcox CJ, Morris G, Entwistle K, Wolfenson D. Effect of season on follicular dynamics and plasma concentrations of estradiol-17 progesterone and luteinizing hormone in lactating Holstein cows. Theriogenology 1994; 42:1263–74. Barkema HW, Schukken YH, Lam TJGM, Bei boer ML, Wilmink H, Benedictus G, Brand A. Incidence of clinical mastitis in dairy herds grouped in three categories by bulk milk somatic cell counts. J Dairy Sci 1998; 81:411-9.

PAGE 105

93 Barker AR, Schrick FN, Lewis MJ, Dowlen HH, Oliver SP. In fluence of clinical mastitis during early lactation on reproductive perf ormance of Jersey cows. J Dairy Sci 1998; 81:1285-90. Barros CM, Newton GR, Thatcher WW, Drost M, Plante C, Hansen PJ. The effect of bovine interferonI1 on pregnancy rate in heifers. J Anim Sci 1992; 70:1471-7. Bartolome JA, Melendez P, Kelbert D, Swift K, McHale J, Hernandez J, Silvestre F, Risco CA, Arteche AC, Thatcher WW, Archbald LF. Strategic use of gonadotrophin-releasing hormone (GnRH) to increase pregnancy rate and reduce pregnancy loss in lactating dairy cows s ubjected to synchronization of ovulation and timed insemination. Theriogenology 2005; 63:1026-37. Bauman DE, Currie WB. Partitioning of nut rients during pregnancy and lactation: a review of mechanisms involving homeosta sis and homeorhesis. J Dairy Sci 1980; 63:1514–29. Beam SW, Butler WR. Energy balance and ovari an follicle development prior to the first ovulation in dairy cows receiving three leve ls of dietary fat. Biol Reprod 1997; 56:133–42 Beam SW, Butler WR. Energy balance, me tabolic hormones, and early postpartum follicular development in dairy cows fe d prilled lipid. J Dairy Sci 1998; 81:121-31. Beam SW, Butler WR. Effects of energy ba lance on follicular development and first ovulation in postpartum dairy cows J Reprod Fertil 1999; 54: 411-24. Berman A, Folman Y, Kaim M, Mamen M, He rz Z, Wolfenson D, Arieli A, Graber Y. Upper critical temperatures and forced ve ntilation effects for high-yielding dairy cows in a subtropical environm ent. J Dairy Sci 1985; 68:1488-95. Bertolini M, Beam SW, Shim H, Bertolin i LR, Moyer AL, Famula TR, Anderson GB. Growth, development, and gene expression by in vivoand in vitro-produced day 7 and 16 bovine embryos. Mol Reprod Dev 2002a; 63:318-28. Bertolini M, Mason JB, Beam SW, Carneiro GF, Sween ML, Kominek DJ, Moyer AL, Famula TR, Sainz RD, Anderson GB. Morphology and morphometry of in vivoand in vitro-produced bovine concepti from early pregnancy to term and association with high birth weig hts. Theriogenology 2002b; 58:973-94. Biggers BG, Geisert RD, Wetteman RP, Buchan an DS. Effects of heat stress on early embryonic development in the beef cow. J Anim Sci 1987; 64:1512-18. Bilby TR, Guzeloglu A, Kamimura S, Pancarci SM, Michel F, Head HH, Thatcher WW. Pregnancy and bovine somatotropin in nonlactating dairy cows: I. Ovarian, conceptus, and insulin-like growth fact or system responses. J Dairy Sci 2004; 87:3256-67.

PAGE 106

94 Binelli M, Thatcher WW, Mattos R, Baruselli PS. Antiluteolytic strategies to improve fertility in cattle. Ther iogenology 2001; 56:1451-63. Block J, Drost M, Monson RL, Rutledge JJ, Rivera RM, Paula-Lopes FF, Ocon OM, Krininger CE III, Liu J, Hansen PJ. Us e of insulin-like growth factor-I during embryo culture and treatment of recipients with gonadotropin-releasing hormone to increase pregnancy rates following the tran sfer of in vitro-produced embryos to heat-stressed, lactating cows J Anim Sci 2003; 81:1590-602. Bo GA, Baruselli PS, Moreno D, Cutaia L, C accia M, Tribulo R, Tribulo H, Mapletoft RJ. The control of follicular wave deve lopment for self-appointed embryo transfer programs in cattle. Theriogenology 2002; 57:53-72. Boyd LJ, Seath DM, Olds D. Relationship be tween level of milk production and breeding efficiency in dairy catt le. J Dairy Sci 1954; 13:89. Breuel KF, Spitzer JC, Henricks DM. Sy stemic progesterone concentration following human chorionic gonadotropin administration at various times during the estrous cycle in beef heifers. J Anim Sci 1989; 67:1564-72. Breukink HJ, Wensing TH. Pathophysiology of the liver in high yielding dairy cows and its consequences for health and produ ction. Bovine Practitioner 1998; 32:74-8. Britt JH, Scott RG, Armstrong JD, Whitacre MD. Determinants of estrous behavior in lactating Holstein cows. J Dairy Sci 1986; 69:2195-202. Bruckental I, Dori D, Kaim M, Lehrer H, Folman Y. Effects of source and level of protein on milk yield and reproductive pe rformance of high-producing primiparous and multiparous dairy cows. Anim Prod 1989; 48:319–29. Bulman DC, Lamming GE. Milk progesterone levels in relation to conception, repeat breeding and factors influe ncing acyclicity in dairy cows. J Reprod Fertil 1978; 54:447-58. Burke JM, Staples CR, Risco CA, De La Sota RL, Thatcher WW. Effect of ruminant grade menhaden fish meal on reproductive and productive performance of lactating dairy cows. J Dairy Sci 1996; 80:3386–98. Butler WR. Nutritional interac tions with reproductive perfor mance in dairy cattle. Anim Reprod Sci. 2000; 60:449-57. Butler WR. Review: effect of protein nutri tion on ovarian and uterine physiology in dairy cattle. J. Dairy Sci. 1998; 81:2533–39. Butler WR, Smith RD. Inte rrelationships between en ergy balance on postpartum reproductive function in dairy ca ttle. J Dairy Sci 1989; 7:767–83.

PAGE 107

95 Carlson JC, Wu XM, Sawada M. Oxygen radi cals and the control of ovarian corpus luteum function. Free Radic Biol Med 1993; 14:79-84. Chassagne M, Barnouin J, Chacorn ac JP. Biological predictors for early clinical mastitis occurrence in Holstein cows under field conditions in France. Prev Vet Med 1998; 16:29-38. Chase CC, Kirby CJ, Hammond AC, Olson TA, Lucy MC. Patterns of ovarian growth and development in cattle with a growth hormone receptor deficiency. J Anim Sci 1998; 76:212-9. Chebel RC, Santos JE, Reynolds JP, Cerri RL, Juchem SO, Overton M. Factors affecting conception rate after artificial inseminati on and pregnancy loss in lactating dairy cows. Anim Reprod Sci 2004; 84:239-55. Chenault JR, Kratzer DD, Rzepkowsky RA, Goodwin MC. LH and FSH response of Holstein heifers to fertirelin acetate, gonadorelin and buser elin. Theriogenology 1990; 34:81-98. Cole JA, Hansen PJ. Effects of administ ration of recombinant bovine somatotropin on the responses of l actating and nonlactating cows to heat stress. J Am Vet Med Assoc 1993; 203:113-7. Crosier A, Farin PW, Dykstra MJ, Alexander JE, Farin CE. Ultras tructural morphometry of bovine blastocysts produced in vivo or in vitro. Biol Reprod 2001; 64:1375-85. Currie EJ. The influence of milk yield on fertility in dairy cattle. J Dairy Sci 1956; 23:301-9. De la Sota RL, Lucy MC, Staples CR, Th atcher WW. Effects of recombinant bovine somatotropin (sometribove) on ovarian func tion in lactating and nonlactating dairy cows. J Dairy Sci 1993; 76:1002-13. De La Torre-Sanchez J, Gardner D, Preis K, Seidel Jr. G. Regulation of glucose metabolism to decrease lipid content of in vitro-produced bovine embryos. Reprod Fertil Dev 2005; 218 [Abstract]. Del Campo MR, Rowe RF, French LR, Ginthe r OJ. Unilateral relationship of embryos and the corpus luteum in cattl e. Biol Reprod 1977; 16:580-5. Del Campo MR, Rowe RF, Chaichareon D, Gint her OJ. Effect of the relative locations of embryo and corpus luteum on embryo su rvival in cattle. Reprod Nutr Dev 1983; 23:303-8. Deluyker HA, Gay JM, Weaver LD, Azari AS. Change of milk yield with clinical diseases for a high producing dair y herd. J Dairy Sci 1991; 74:436-45.

PAGE 108

96 de Vries A, Risco CA. Trends and seasona lity of reproductive performance in Florida and Georgia dairy herds from 1976 to 2002. J Dairy Sci 2005; 88:3155-65. Diaz T, Schmitt EJ, de la Sota RL, Thatch er MJ, Thatcher WW. Human chorionic gonadotropin-induced alterations in ovarian follicular dynamic s during the estrous cycle of heifers. J Anim Sci 1998; 76:1929-36. Diez C, Heyman Y, Le Bourhis D, Guya der-Joly C, Degrouard J, Renard, JP. Delipidating in vitro-produced bovine z ygotes: Effect on further development and consequences for freezability. Theriogenology 2001; 55: 923-36. Dinnys A, Wallace GA, Rall WF. Effect of genotype on the efficiency of mouse embryo cryopreservation by vitrification or slow freezing methods. Mol Reprod Dev 1995; 40:429-35. Djemali M, Berger PJ, Freeman AE. Ordered categorical sire evaluation for dystocia in Holsteins. J Dairy Sci 1987; 70:2374-84. Dobrinsky JR, Overstrm EW, Duby RT, J ohnson LA, Duffy P, Roche JF, Boland MP. Effect of cytoskeletal stabilization on the development of bovine embryos cryopreserved by vitrification. Ther iogenology 1995; 43:199 (Abstract). Dobrinsky JR. Cellular approach to cryopr eservation of embryos. Theriogenology 1996; 45:17-26. Dobrinsky JR, Pursel VG, Long CR, Johnson LA Birth of piglets after transfer of embryos cryopreserved by cytoskeletal stab ilization and vitrification. Biol Reprod 2000; 62:564-70. Donaghy AJ, Baxter RC. Insulin-like growth factor bioactivity and its modification in growth hormone resistant states. Baillie res Clin Endocrinol Metabol 1996; 10:421– 446. Donovan DC, Schingoethe DJ, Baer RJ, Ryali J, Hippen AR, Franklin ST. Influence of dietary fish oil on conjugated linoleic acid and other fatty acids in milk fat from lactating dairy cows. J Dairy Sci 2000;83: 2620-8. Dransfield MB, Nebel RL, Pearson RE, Warn ick LD. Timing of insemination for dairy cows identified in estrus by a radiotelem etric estrus detection system. J Dairy Sci 1998; 81:1874-82. Drost M, Thatcher JD, Cantrell CK, Wolf sdorf KE, Hasler JF, Thatcher WW. Conception rates after artificial inseminati on or transfer of frozen/thawed embryos to lactating cows during summer. J Dairy Sci 1994; 77:380 (Abstract). Drew SB, Peters AR. Effect of buserelin on pregnancy rates in dairy cows. Vet Rec 1994; 134:267–69.

PAGE 109

97 Dunlap SE, Vincent CK. Influence of postb reeding thermal stress on conception rate in beef cattle. J Anim Sci 1971; 32: 1216-8. Ealy AD, Drost M, Hansen PJ. Developmental changes in embryonic resistance to adverse effects of maternal heat st ress in cows. J Dairy Sci 1993; 76:2899–905. Ealy AD, Arechiga CF, Bray DR, Risco CA, Ha nsen PJ. Effectiveness of short-term cooling and vitamin E for alleviation of in fertility induced by heat stress in dairy cows. J Dairy Sci 1994; 77:3601-7. Ealy AD, Hansen PJ. Induced thermotolera nce during early development of murine and bovine embryos. J Cell Physiol 1994; 160:463-8. Ealy AD, Howell JL, Monterroso VH, Arechiga CF, Hansen PJ. Developmental changes in sensitivity of bovine embryos to h eat shock and use of antioxidants as hermoprotectants. J Anim Sci 1995; 73:1401-7. Ealy A D, Drost M, Barros CM, Hansen PJ. Thermoprotection of pr eimplantation bovine embryos from heat shock by glutathione and taurine. Cell Biol Int Rep 1992; 16:125 [abstract]. Echternkamp SE, Gregory KE, Dickerson GE, Cundiff LV, Koch RM, Van Vleck LD. Twinning in cattle: II. Genetic and envi ronmental effects on ovulation rate in puberal heifers and postpartum cows and th e effects of ovulation rate on embryonic survival. J Anim Sci 1990; 68:1877-88. Edwards JL, Hansen PJ. Differential respons es of bovine oocytes and preimplantation embryos to heat shock. Mol Reprod Dev 1997; 46:138-145. Enright BP, Lonergan P, Dinnyes A, Fair T, Ward FA, Yang X, Boland MP. Culture of in vitro produced bovine zygot es in vitro vs in vivo: Implications for early embryo development and quality. Theriogenology 2000; 54:659-73. Erickson PJ, Murphy MR, Clark JH. Supplemen tation of dairy cow diets with calcium salts of longchain fatty acids and nicotinic acid in early lacta tion. J Dairy Sci 1992; 75:1078–89. Fahy GM, MacFarlane DR, Angell CA, Meryman HT. Vitrification as an approach to cryopreservation. Cryobiology 1984; 21:407-26. Fair T, Lonergan P, Dinnyes A, Cotell DC, Hyttel P, Ward FA, Boland MP. Ultrastructure of bovine bl astocysts following cryopreser vation: Effects of method of blastocyst production. Mo l Reprod Dev 2001; 58: 186-95. Falconer DS. 1981. Introduction to Quantitati ve Genetics. 2nd ed. Longman House, Harlow, Essex, UK.

PAGE 110

98 Farin CE, Farin PW, Piedrahita JA. Developm ent of fetuses from in vitro-produced and cloned bovine embryos. J Anim Sci 2004; 82:53-62. Farin PW, Piedrahita JA, Farin CE. Errors in development of fetuses and placentas from in vitro-produced bovine embryos. Theriogenology 2006; 30 (in press). Faust MA, McDaniel AB, Robinson OW, Britt JH. Environmental and yield effects on reproduction in primiparous Hols teins. J Dairy Sci 1988; 71:3092. Ferguson J D, Sklan D, Chalupa WV, Kronfeld DS. Effects of hard fats on in vitro and in vivo rumen fermentation, milk production, and reproduction in dairy cows. J Dairy Sci 1990; 73:2864–79. Fischer-Brown A, Monson R, Parrish J, Rutle dge J. Cell allocation in bovine embryos cultured in two media under two oxygen concentrations, Zygote 2002; 10:341–8. Fleischer P, Metzner M, Beyerbach M, Ho edemaker M, Klee W. The relationship between milk yield and the incidence of so me diseases in dairy cows. J Dairy Sci 2001; 84:2025-35. Fonseca FA, Britt JH, McDaniel BT, Wilk JC Rakes AH. Reproductive traits of Holsteins and Jerseys. Effects of age, milk yield, and clinical abnormalities on involution of cervix and ut erus, ovulation, estrous cycl es, detection of estrus, conception rate, and days open. J Dairy Sci 1983; 66:1128-47. Fourichon C, Seegers H, Malher X. Effect of disease on reproduction in the dairy cow: a meta-analysis. Theriogenology 2000; 53:1729–1759. Fujitani Y, Kasai K, Ohtani S, Nishimura K, Yamada M, Utsumi K. Effect of oxygen concentration and free radical s on in vitro development of in vitro-produced bovine embryos. J Anim Sci 1997; 75:483-9. Gaines Wl. Milk yield in relation to recurrence of con ception. J Dairy Sci 1927; 10:117. Garcia-Bojalil CM, Staples CR, Risco CA Savio JD, Thatcher WW. Protein degradability and calcium salts of long-chai n fatty acids in the diets of lactating dairy cows: reproductive responses J Dairy Sci 1998; 81:1385-95. Garcia-Bojalil CM. 1993. Reproductive, pr oductive, and immunological responses by Holstein dairy cows fed diets varying in concentration and rumi nal degradability of protein and supplemented with ruminally-i nert fat. Ph.D. Diss., Univ. Florida, Gainesville. Garrett JE, Geisert RD, Zavy MT, Morgan GL. Evidence for maternal regulation of early conceptus growth and development in beef cattle Reprod Fertil 1988a; 84:437–46.

PAGE 111

99 Garrett JE. Geisert RD. Zavy NIT, Gries LK Wetteman RP. Buchanan DS. Effect of exogenous progesterone on prostaglandin F2 release and the interestrous interval in the bovine. Prostaglandins 1988b; 36:85-96. Ginger, R., Faisser, D., Busato, A., Blum, J. and Kupfer, U. Blood parameters during early lactation and their relationship to ovarian function in dairy cows. Reprod Domest Anim 1997; 32:313-9. Ginther OJ. Effect of progesterone on lengt h of estrous cvcle in cattle. Am J Vet Res 1970; 31:493-6. Ginther OJ, Knopf L, Kastelic JP. Temporal associations among ovarian events in cattle during oestrus cycles with two and three follicular waves. J Reprod Fertil 1989; 87:223-30. Ginther OJ, Wiltbank MC, Fricke PM, Gibbons JR, Kot K. Selection of the dominant follicle in cattle. Biol Reprod 1996; 55:1187–94. Gong JG, Bramley TA, Webb R. The effect of recombinant bovine somatotrophin on ovarian follicular growth and development in heifers. J Reprod Fertil 1993; 97:24754. Gossen N, Feldmann M, Hoedemaker M. [Effect of parenteral supplementation with carotene in the form of an injection solution (Carofertin) on the fertility performance of dairy cows] Dtsch Ti erarztl Wochenschr 2004;111:14-21. Gossen N, Hoedemaker M.[Effect of beta-carotin serum concentration on the reproductive performance in dairy cows] Berl Munch Tierarztl Wochenschr 2005; 118:326-33. Green LE, Hedges VJ, Schukken YH, Blowey RW, Packington AJ. The impact of clinical lameness on the milk yield of dairy cows. J Dairy Sci 2002; 85:2250-6. Gr hn YT, Wilson DJ, Gonzalez RN, Hertl JA Schulte H, Bennett G, Schukken YH. Effect of pathogen-specific clinical mas titis on milk yield in dairy cows. J Dairy Sci 2004; 87:3358-74. Grummer RR, Carroll DJ. Effects of dietary fat on metabolic disorders and reproductive performance of dairy cattle. J Anim Sci 1991; 69:3838–52 Guyader-Joly C, Ponchon S, Durand M, Heyman Y, Renard JP, Mnzo Y. Effect of lecithin on in vitro and in vivo survival of in vitro produced bovine blastocysts after cryopreservation. Theri ogenology 1999; 52:1193-202. Gwazdauskas FC, Thatcher WW, Kiddy CA, Paape MJ, Wilcox, CJ. Hormonal pattern during heat stress following tham salt i nduced luteal regression in heifers. Theriogenology 1981; 16:271–285.

PAGE 112

100 Hansen L, Heins B, Seykora T. Crossbreed ing: Why the interest? What to expect. Proceedings Florida Dairy Production C onference, Gainesville, May 3, 2005: 42:14-20. Hansen LB. Consequences of selection for milk yield from a geneticist's viewpoint. J Dairy Sci 2000; 83:1145-50. Hansen PJ, Archiga CF. Strategies for ma naging reproduction in th e heat-stressed dairy cow. J. Anim. Sci. 1999; 77:36-50. Hansen PJ, Drost M, Rivera RM, Paula-L opes FF, Al-Katanani YM, Krininger III CE, Chase CC Jr. Adverse impact of heat stress on embryo production: Causes and strategies for mitigation. Theriogenology 2001; 55:91-103. Hansen PJ, Block J. Towards an embryocentric world: the current a nd potential uses of embryo technologies in dairy producti on. Reprod Fertil Dev 2004; 16:1-14. Hansel W. Plasma hormone concentrations associated with early embryo mortality in heifers. J Reprod Fe rtil 1981; 30:231–9. Hardingham TE, Fosang AJ. Proteoglycans: many forms and many functions. FASEB J 1992; 6:861-70. Harrison JH, Hancock DD, St Pi erre N, Conrad HR, Harvey WR. Effect of prepartum selenium treatment on uterine involution in the dairy cow. J Dairy Sci 1986; 69:1421-5. Harrison RO, Ford SP, Young JW, Conley AJ Freeman AE. Increased milk production versus reproductive and energy status of high producing dairy cows. J Dairy Sci 1990; 73:2749-58. Hasler JF, Henderson WB, Hurtgen PJ, Jin ZQ, McCauley AD, Mower SA, Neely B, Skuey LS, Stokes, JE, Trimmer SA. Pr oduction, freezing and transfer of bovine IVF embryos and subsequent calving results. Theriogenology 1995; 43:141-52. Hasler, JF, Hurtgen PJ, Jin ZQ, Stokes JE. Survival of IVF-derived bovine embryos frozen in glycerol or ethylene glycol. Therioge nology 1997; 48:563-79. Hasler JF. In-vitro production of cattle embryos: problems with pregnancies and parturition. Hum Re prod 2000; 15:47-58. Hasler, JF. The current status and future of commercial embryo transfer in cattle. Anim Reprod Sci 2003a; 79:245-64. Hasler JF, Bilby CR, Collier RJ, Denham SC, Lucy MC. Effect of recombinant bovine somatotropin on superovulatory response and recipient pregnancy rates in a commercial embryo transfer progr am. Theriogenology 2003b; 59:1919-28.

PAGE 113

101 Hawkins DE, Niswender KD, Oss GM, Moelle r CL, Odde KG, Sawyer HR, Niswender GD. An increase in serum lipids increases luteal lipid content and alters the disappearance rate of pr ogesterone in cows. J Anim Sci 1995; 73:541–5. Heersche Jr G, Nebel RL. M easuring efficiency and accuracy of detection of estrus. J Dairy Sci 1994; 77:2754–61. Hernandez J, Shearer JK, Webb DW. Eff ect of lameness on the calving-to-conception interval in dairy cows. J Am Vet Med Assoc 2001; 15:1611-4. Heyman Y, Chesn P, Chupin D, Mnzo Y. Improvement of survival rate of frozen cattle blastocysts after tran sfer with trophoblastic vesi cles. Theriogenology 1987; 27:477-84. Hillers JK, Senger PL, Darlington RL, Flem ing WN. Effects of production, season, age of cow, days dry, and days in milk on conception to first service in large commercial dairy herds. J Dairy Sci 1984; 67:861. Hommeida A, Nakao T, Kubota H. Luteal function and conc eption in lactating cows and some factors influencing luteal functi on after first insemination. Theriogenology 2004; 62:217-25. Howell JL, Fuquay JW, Smith AE. Corpus lu teum growth and function in lactating Holstein cows during spring a nd summer. J Dairy Sci 1994; 77:735–9. Ikeda K, Takahashi Y, Katagiri S. Effect of medium change on the development of in vitro matured and fertilized bovine oocyte s cultured in medium containing amino acids. J Vet Med Sci 2000; 62:121-3. Ingraham RH, Gillette DD, Wagner WD. Rela tionship of temperature and humidity to conception rate of Holstein cows in subtropical climate. J Dairy Sci 1974;57:47681. Inskeep EK. Factors that affect fertility duri ng estrous cycles with s hort luteal phases in postpartum cows. J Repr od Fertil 1995; 49: 493–503. Ireland JJ, Roche JF. Effect of progester one on basal LH and episodic LH and FSH secretion in heifers. J Reprod Fertil 1982;64:295-302. Ishii T, Matsuki S, Iuchi Y, Okada F, Toyosaki S, Tomita Y, Ikeda Y, Fujii J. Accelerated impairment of spermatogeni c cells in SOD1-knockout mice under heat stress. Free Radic Res 2005; 39:697-705. Iwasaki S, Hamano S, Kuwa yama M, Ushijima H, Nagaoka S, Nakahara T. Developmental changes in the inciden ce of chromosomal anomalies of bovine embryos fertilized in vitro. J Exp Zool 1992; 261:79-85.

PAGE 114

102 Jang G, Lee BC, Kang SK, Hwang WS. E ffect of glycosaminoglycans on the preimplantation development of embryos de rived from in vitro fertilization and somatic cell nuclear transfer. Re prod Fertil Dev 2003; 30:179-85. Jones JI, Clemmons DR. In sulin-like growth factors and their binding proteins: biological actions. Endocrine Reviews 1995; 16:3–34. Jorritsma R, Jorritsma H, Schukken YH, Wentin k GH. Relationships between fatty liver and fertility and some periparturient dis eases in commercial Dutch dairy herds. Theriogenology 2000; 54:1065–74. Jousan FD, Drost M, Hansen PJ. Factors asso ciated with early and mid-to-late fetal loss in lactating and non-lactati ng Holstein cattle in a hot climate. J Anim Sci 2005; 83:1017-22. Jubb TF, Abhayaratne D, Malmo J, Anderson GA. Failure of an in tramuscular injection of an analogue of GnRH 11 to 13 days after insemination to increase pregnancy rates in dairy cattle. Aust Vet J 1990; 67:359–61. Juengel JL, Garverick HA, Johnson AL, Youngq uist RS, Smith MF. Apoptosis during luteal regression in cattle. Endocrinology 1993; 132:249–54. Kadarmideen HN, Thompson R, Simm G. Linear and threshold model genetic parameter estimates for disease, fertility and producti on traits in UK dairy cattle. J Anim Sci 2000; 71:411–9. Kappel LC, Ingraham RH, Morgan EB, Dixon JM, Zeringue L, Wilson D, Babcock DK. Selenium concentrations in feeds and eff ects of treating pregnant Holstein cows with selenium and vitamin E on bloo d selenium values and reproductive performance. Am J Vet Res 1984;45:691-4. Kelton DF, K D Lissemore, Martin RE. Reco mmendations for recording and calculating the incidence of selected clinical diseases of dair y cattle. J Dairy Sci 1998; 81:2502–9. Kerbler TL, Buhr MM, Jordan LT, Leslie KE Walton JS. Relationship between maternal plasma progesterone concen tration and interferonsynthesis by the conceptus in cattle. Theriogenology 1997; 47:703–14. Khurana N, Niemann, H. Effects of cryopreservation on glucose metabolism and survival of bovine morulae and blasto cysts derived in vitro or in vivo. Theriogenology 2000a; 54:313-26. Khurana NK, Niemann H. Energy metabo lism in preimplantation bovine embryos derived in vitro or in vi vo. Biol. Reprod 2000b; 62:847-56.

PAGE 115

103 Kim H, Lee G, Hyun S, Nam D, Lee S, Jeong Y, Kim S, Kim J, Kang S, Lee B, Hwang W. Embryotropic effect of glycosaminogl ycans and receptors in development of porcine pre-implantation embryos. Theriogenology 2005; 63:1167-80. Kimura M, Nakao T, Moriyoshi M, Kawata K. Luteal pha se deficiency as a possible cause of repeat breeding in da iry cattle. Br Vet J 1987; 143:560–6. Kimura K, Spate LD, Green MP, Roberts RM Effects of D-glucose concentration, Dfructose, and inhibitors of enzymes of the pentose phosphate pathway on the development and sex ratio of bovine bl astocysts. Mol Reprod Dev 2005; 72:201-7. Kinsel ML, Marsh WE, Ruegg PL Etherington WG. Risk f actors for twinning in dairy cows. J Dairy Sci 1998; 81:989-93. Knopf L, Kastelic JP, Schallenberger E, Gi nther OJ. Ovarian follicular dynamics in heifers: Test of two-wave hypothesis by ultrasonically monitoring individual follicles. Domest Anim Endocrinol 1989; 6:111-19. Ko JCH, Kastelic JP, Del Campo MR, Ginthe r OJ. Effects of a dominant follicle on ovarian follicular dynamics during the oestro us cycle in heifers. J Reprod Fertil 1991; 91:511-19. Krisher RL, Lane M, Bavister BD. De velopmental competence and metabolism of bovine embryos cultured in semi-defined and defined culture media. Biol Reprod 1999; 60:1345-52. Kruip TAM, den Daas JHG. In vitr o produced and cloned embryos: Effects on pregnancy, parturition and offs pring. Theriogenology 1997; 47:43–52. Kuwayama M, Fujikawa S, Nagai T. Ultr astructure of IVM-IV F bovine blastocysts vitrified after equilibration in glycerol 1,2-propanediol using 2-step and 16-step procedures. Cryobiology 1994; 31:415-22. Laben RC, Shanks RD, Berger PJ, Freeman AE. Factors affecting milk yield and reproductive performance. J Dairy Sci 1982; 65:1004. Lafrance M, Goff AK. Effects of progester one and oestradiol17 beta on oxytocininduced release of prostaglandin F2 in heifers. J Reprod Fertil 1988; 82:429-36. Lajili H, Humblot P, Thibier M. Effects of PGF2 alpha treatment on conception rates of dairy cows treated with a GnRH agonist 12 to 14 days after ar tificial insemination. Theriogenology 1991; 36:335-47. Lamming GE, Darwash AO. Effect of inter-lute al interval on subsequent luteal phase length and fertility in postpartun dairy cows. Biol Reprod 1995; 53:63.

PAGE 116

104 Lammoglia MA, Willard ST, Hallford DM and Randel RD. Effects of dietary fat on follicular development and circulating concentrations of lipids, insulin, progesterone, estradiol 17b, 13,14dihydro-15-ketopr ostaglandin F2 and growth hormone in estrous cyclic Brah man cows. J Anim Sci 1997; 75:1591–600 Lane M, Maybach JM, Hooper K, Hasler JF, Gardner DK. Cryo-survival and development of bovine blastocysts are enhanced by culture with recombinant albumin and hyaluronan. Mol Reprod Dev 2003; 64:70-78. Larson SF, Butler WR, Currie WB. Reduced fe rtility associated with low progesterone postbreeding and increased milk urea nitr ogen in lactating co ws. J Dairy Sci 1997; 80:1288-95. Laurent TC. Biochemistry of hyalu ronan. Acta Otolar yngol 1987; 442:7-24. Lazzari G, Wrezycki C, Herrmann D, Duchi R, Kruip T, Niemann H, Galli C. Cellular and molecular deviations in bovine in v itro-produced embryos are related to the large offspring syndrome. Biol Reprod 2002; 67:767-75. Lee CN, Ax RL. Concentration and compos ition of glycosaminoglycans in the female bovine reproductive tract. J Dairy Sci 1984; 67:2006-9. Lemaster JW, Seals RC, Hopkins FM, Schrick FN. Effects of administration of oxytocin on embryonic survival in progestogen suppl emented cattle. Prostaglandins Other Lipid Mediat 1999; 57:259-68. Leeuwenberg BR, Hudson NL, Moore LG, Hurst PR, McNatty KP. Peripheral and ovarian IGF-I concentrations during the ovine oestrous cycl e. J Endocrinol 1996; 148: 281-9. Levine L, Worth N. Eicosapentaenoic acid – its effects on arach idonic acid metabolism by cells in culture. J Allergy and Clin Immun 1984; 74:430–6. Loeffler SH, de Vries MJ, Schukken YH. The e ffects of time of dis ease occurrence, milk yield, and body condition on fertility of dairy cows. J Dairy Sci 1999; 82:2589-604. Lonergan P, Rizos D, Gutierrez-Adan A, Moreira PM, Pintado B, De La Fuente J, Boland MP. Temporal divergence in the patter n of messenger RNA expression in bovine embryos cultured from the zygote to blasto cyst stage in vitro or in vivo. Biol Reprod 2003; 69:1424-31. Lpez-Gatius F. Is fertility declining in dairy cattle? A retrospective study in northeastern Spain. Th eriogenology 2003;60:89-99. Lpez-Gatius F, Santolaria P, Yaniz J, Fen ech M, Lopez-Bejar M. Risk factors for postpartum ovarian cysts and their spontane ous recovery orpersis tence in lactating dairy cows. Theriogenology. 2002; 58:1623-32.

PAGE 117

105 Lpez-Gatius F, Hunter RH. Spontaneous reduction of advanced twin embryos: its occurrence and clinical re levance in dairy cattle. Theriogenology 2005; 63:118-25. Lpez-Gatius F, Santolaria P, Martino A, Delta ng F, De Rensis F. The effects of GnRH treatment at the time of AI and 12 days later on reproductive performance of high producing dairy cows during the wa rm season in northeastern Spain. Theriogenology 2005a; (in press). Lpez-Gatius F, Lopez-Bejar M, Fenech M, Hunter RH. Ovulation failure and double ovulation in dairy cattle: risk factor s and effects. Theriogenology 2005b; 63:1298307. Lpez H, Satter LD, Wiltbank MC. Relations hip between level of milk production and estrous behavior of lactating dairy cows. Anim Reprod Sci 2004; 81:209-23. Lpez H, Caraviello DZ, Satter LD, Fricke PM, Wiltbank MC. Relationship between level of milk production and multiple ovulat ions in lactating dairy cows. J Dairy Sci 2005; 88:2783-93. Loucopoulos A, Ferin M. Gonadotropin-releas ing hormone and its clinical applications. Obstet Gynecol Annu 1984; 13:275-88. Lucy MC. Reproductive loss in high-producing dairy cattle: where will it end? J Dairy Sci. 2001; 84:1277-93. Lucy MC, Berk J, Staples CR, Head HH, Sota RLD, Thatcher WW. Follicular dynamics, plasma metabolites, hormones and insulin-lik e growth factor I (IG F-1) in lactating cows with positive or negative energy balance during the preovulatory period. Reprod Nutr Dev 1992a, 32:331-41. Lucy MC, De la Sota RL, Staples CR, That cher WW. Ovarian follicular populations in lactating dairy cows treated with recomb inant bovine somatotropin (sometribove) or saline and fed diets differing in fat content and energy. J Dairy Sci 1993; 76:1014–27. Lucy MC, Gross TS, Thatcher WW. Effect of intravenous infusion of soybean oil emulsion on plasma concentrations of 15-keto-13,14dihydro-prostaglandin F2 and ovarian function in cycling Holstein heifers. In International Atomic Energy Agency Livestock Reproduction in Latin America 1990; 119–32. Lucy MC, Staples CR, Michel FM, Thatcher WW. Effect of feeding calcium soaps to early post-partum dairy cows on plasma prostaglandin F2 luteinizing hormone, and follicular growth. J Dairy Sci 1991a; 74:483–9. Lucy MC, Staples CR, Michel FM, Thatcher WW. Energy balance and size and number of ovarian follicles detected by ultrasonogr aphy in early post-partum cows. J Dairy Sci 1991b; 74:473–82.

PAGE 118

106 Lucy MC, Staples CR, Thatcher WW, Eric kson PS, Cleale RM, Firkins JL, Clark JH, Murphy MR, Brodie BO. Influence of diet composition, dry matter intake, milk production and energy balance on time of postpartum ovulation and fertility in dairy cows. Anim Prod 1992b; 54:323–31. Lucy MC, Stevenson JS. Gonadotropin-re leasing hormone at estrus: luteinizing hormone, estradiol, and progesterone dur ing the periestrual and postinsemination periods in dairy cattle. Bi ol Reprod 1986; 35:300-11. Lucy MC, Weber WJ, Baumgard LH, Seguin BS, Koenigsfeld AT, Hansen LB. Reproductive endocrinology of lactating dair y cows selected fo r increased milk production. J. Dairy Sci 1998; 81:246 (abstract). Lukaszewska J, Hansel W. Corpus luteum maintenance during ear ly pregnancy in the cow. J Reprod Fertil 1980; 59:485-93. Lynch PR, Macmillan KL, Taufa VK. Treatin g cattle with progesterone as well as a GnRH analogue affects oestrous cycle le ngth and fertility. Anim Reprod Sci 1999; 56:189-200. Macmillan KL, Taufa VK, Day AM. Effects of an agonist of GnRH (buserelin) in cattle. III. Pregnancy rates after a post-insemination injection during metoestrus or dioestrus. Anim Reprod Sci 1986; 11:1–10. Macmillan KL, Thatcher WW. Effects of an agonist of gonadotropin-releasing hormone on ovarian follicles in cattle. Biol Reprod 1991; 45:883-9. Mahanna WC, Hardie AR, Tyler WJ. Variati on in health related expenditures of Holstein-Friesian cattle. J Daiy Sci 1979; 62:86 (abstract). Malayer JR, Hansen PJ. Differences between Brahman and Holstein cows in heat-shock induced alterations of protein synthesi s and secretion by oviducts and uterine endometrium. J Anim Sci 1990; 68:266-80. Mann GE, Lamming GE. Effects of treatment with buserelin on plasma concentrations of oestradiol and progesterone and cy cle length in the cow. Br Vet J 1995a; 151:427-32. Mann GE, Lamming GE. Progesterone inhibition of the development of the luteolytic signal in cows. J Repr od Fertil 1995b; 104:1–5. Mann GE, Mann SL, Lamming GE. The inte r-relationship between the maternal hormone environment and the embryo during the early stages of pregnancy in the cow. J Reprod Fertil 1996; 17:21. Mann GE, Lamming GE, Payne JH. Role of early luteal phase progesterone in control of the timing of the luteolytic si gnal. J Reprod Fertil 1998; 113:47–51.

PAGE 119

107 Mann GE, Lamming GE. The influence of proge sterone during early pregnancy in cattle. Reprod Domest Anim 1999; 34:269-74. Mann GE, Lamming GE. Relationship between maternal endocrine environment, early embryo development and inhibition of lute olytic mechanism in cows. Reproduction 2001; 121:175–80. Margolin Y, Behrman HR. Xanthine oxidase and dehydrogenase activities in rat ovarian tissues. Am J Physiol 1992; 262:173-8. Martinez MF, Kastelic JP, Adams GP, Cook RB, Olson WO, Mapletoft RJ. The use of progestins in regimens for fixed-time ar tificial insemination in beef cattle. Theriogenology 2002b; 57:1049–59. Martinez MF, Kastelic JP, Adams GP, Mapletof t RJ. The use of a progesterone-releasing device (CIDR-B) or melengest rol acetate with GnRH, LH, or estradiol benzoate for fixed-time AI in beef heifers. J Anim Sci. 2002a; 80:1746-51. Martino A, Songsasen N, Leibo SP. Devel opment into blastocyst s of bovine oocytes cryopreserved by ultra-rapid coolin g. Biol Reprod 1996a; 54:1059-69. Martino A, Pollard JW, Leibo SP. Eff ect of chilling bovine oocytes on their developmental competence. Mol Reprod Dev 1996b; 45:503-12. Massip A, Mermillod P, Wils C, Dessy F. Effects of dilution procedure and culture conditions after thawing on survival of fr ozen bovine blastocysts produced in vitro. J Reprod Fertil 1993; 97:65-9. Massip A, Mermillod P, Dinnyes A. Morpholog y and biochemistry of in vitro produced bovine embryos: implications for th eir cryopreservation. Hum Reprod 1995; 10:3004-11. Mattos R, Staples CR, Thatcher WW. Effect s of dietary fatty aci ds on reproduction in ruminants. Rev Reprod 2000; 5:38-45. Mattos R, Guzeloglu A, Thatcher WW. Effect of polyunsaturated fatty acids on secretion of PGF2 from bovine endometrial (BEND) cells stimulated with phorbol 12, 13 dibutyrate (PDBu). Theri ogenology 2001; 55:326. Mattos R, Staples CR, Arteche A, Wiltbank MC, Diaz FJ, Jenkins TC, Thatcher. The effects of feeding fish oil on uterine s ecretion of PGF2alpha, milk composition, and metabolic status of periparturient Ho lstein cows. J Dairy Sci 2004; 87:921-32. McAllister AJ, Lee AJ, Batra TR, Lin CY, R oy GL, Vesely JA, Wauthy JM, Winter KA. The influence of additive and nonadditive gene action on lifetime yields and profitability of da iry cattle. J Dairy Sci 1994; 77:2400-14.

PAGE 120

108 McNatty KP, Heath DA, Lundy T, Filder AE, Quirke L, O’Connell A, Smith P, Groom N, Tisdall DJ. Control of early ovarian follicular development. J Reprod Fertil 1999; 54:3–16. Mee MO, Stevenson JS, Scoby RK, Folman Y. Influence of gonadotropin-releasing hormone and timing of insemination relative to estrus on pregnancy rates of dairy cattle at first service. J Dairy Sci 1990; 73:1500-7. Mee MO, Stevenson JS, Alexander BM, Sasser RG. Administration of GnRH at estrus influences pregnancy rates, serum concentr ations of LH, FSH, estradiol-17 beta, pregnancy-specific protein B, and progesterone, proportion of luteal cell types, and in vitro production of progesterone in dairy cows. J Anim Sci 1993; 71:185-98. Melendez P, Bartolome J, Archbald LF, Donovan A. The association between lameness, ovarian cysts and fertility in lactating dairy cows. Theriogenology 2003; 59:927-37. Meyer MD, Hansen PJ, Thatcher WW, Drost M, Badinga L, Roberts RM, Li J, Ott TL, Bazer FW. Extension of corpus luteum lifespan and reduction of uterine secretion of prostaglandin F2 of cows in response to recombinant interferon. J Dairy Sci 1995; 78:1921-31. Milvae RA, Hansel W. Concurrent uterine venous and ovarian arte rial prostaglandin F2 concentrations in heifers treated w ith oxytocin. J Reprod Fenil 1980; 60:715. Monty DE, Racowsky C. In vitro evaluation of early embryo viability and development in summer heat-stressed, superovulat ed dairy cows. Theriogenology 1987; 28:451465. Morales-Roura JS, Zarco L, Hernandez-Ceron J, Rodriguez G. Ef fect of short-term treatment with bovine somatotropin at estr us on conception rate and luteal function of repeat-breeding dairy cows Theriogenology 2001; 55:1831-41. Moreira F, de la Sota RL, Diaz T, Thatcher WW. Effect of day of th e estrous cycle at the initiation of a timed artificial inseminati on protocol on reproductive responses in dairy heifers. J Anim Sci 2000a; 78:1568-76. Moreira F, Risco CA, Pires MF, Ambrose JD Drost M, Thatcher WW. Use of bovine somatotropin in lactating dairy cows rece iving timed artificial insemination. J Dairy Sci. 2000b; 83:1237-47. Moreira F, Orlandi C, Risc o CA, Mattos R, Lopes F, Thatcher WW. Effects of presynchronization and bovine somatotropin on pregnancy rates to a timed artificial insemination protocol in lactating da iry cows. J Dairy Sci 2001; 84:1646-859. Moreira F, Badinga L, Burnley C, Thatch er WW. Bovine somatotropin increases embryonic development in superovulated cows and improves post-transfer pregnancy rates when given to lactat ing recipient cows. Theriogenology 2002; 57:1371-87.

PAGE 121

109 Murakami M, Otoi T, Sumantri C, Suzuki T. Effects of centrifuga tion and lipid removal on the cryopreservation of in vitro produced bovine embr yos at the eight-cell stage. Cryobiology 1998; 36:206-12. Murphy LJ, Bell GI, Friesen HG. Tissue distri bution of insulin-like growth factor-I and II messenger ribonucleic acid in the adult rat. Endocrinology 1987; 120: 1279-282. Nebel RL, McGilliard ML. Interactions of high milk yield and re productive performance in dairy cows. J Dairy Sci 1993; 76:3257-68. Nebel RL, Jobst SM, Dransfield MBG, Pandolfi SM, Bailey TL. Use of radio frequency data communication system, HeatWatch to describe behavior al estrus in dairy cattle. J Dairy Sci 1997; 80:179 (Abstr.). Nedambale TL, Dinnyes A, Groen W, Dobrin sky JR, Tian XC, Yang X. Comparison on in vitro fertilized bovine embryos cultu red in KSOM or SOF and cryopreserved by slow freezing or vitrification. Theriogenology 2004a; 62:437-49. Nedambale TL, Dinnyes A, Yang X, Tian XC. Bovine blastocyst development in vitro: timing, sex, and viability following vitr ification. Biol Re prod 2004b; 71:1671-6. Newton G R, Martinod S, Hansen PJ, Thatcher WW, Siegenthaler B, Gerber C, Voirol MJ. Effect of bovine interferon on acut e changes in body temperature and serum progesterone concentration in he ifers. J Dairy Sci 1990; 73:3439. Nielen MYH, Schukken DT, Scholl HJ, Wilbrink HJ, Brand A. Twinning in dairy cattle: a study of risk factors and eff ects. Thaiogenology 1989; 32:845. Niki E, Noguchi N, Tsuchihashi H, Gotoh N. Interaction among vitamin C, vitamin E, and beta-carotene. Am J C lin Nutr 1995; 62:1322-6. O’Callaghan DO, Boland MP. Nutritional e ffects on ovulation, embryo development and the establishment of pregnancy in ruminants. J Anim Sci 1999; 68:299–314. O’Connor ML, Senger PL, 1997: Estrus dete ction. In: Youngquist, RS (ed.), Current Therapy in Large Animal Therioge nology, Chapter 34. W.B. Saunders Co., Philadelphia, USA, p. 285. Oldick BS, Staples CR, Thatcher WW, Gyawu P. Abomasal infusion of glucose and fat – effects on digestion, production, and ovari an and uterine func tions of cows. J Dairy Sci 1997; 80:1315–28. Olds D, Cooper T, Thrift FA. Relationships between milk yield and fertility in dairy cattle. J Dairy Sci 1979; 62:1140-4. Palasz AT, Mapletoft RJ. Cryopreservation of mammalian embryos and oocytes: recent advances. Biotechnol Adv 1996; 14:127-49.

PAGE 122

110 Parrish JJ, Susko-Parrish JL, Critser ES, Ey estone WH, First NL. Bovine in vitro fertilization with frozen-thawe d semen. Theriogenology 1986; 25:591-600. Paula-Lopes FF, Al-Katanani YM, Maje wski AC, McDowell LR, Hansen PJ. Manipulation of antioxidant status fails to improve fertility of lactating cows or survival of heat-shocked embr yos. J Dairy Sci 2003; 86:2343-51. Pecsok SR, McGilliard ML, Nebel RL. Concep tion rates. 1. Derivation and estimates for effects of estrus detection on cow pr ofitability. J Dairy Sci 1994; 77:3008-15. Pell JM, Saunders JC, Gilmour RS. Differen tial regulation of transcription initiation from insulin-like growth factor-I (IG F-I) leader exons a nd of tissue IGF-I expression in response to changed growth hormone and nutritional status in sheep. Endocrinology 1993; 132:1797–1807. Perry GA, Smith MF, Lucy MC, Green JA, Parks TE, MacNeil MD, Roberts AJ, Geary TW. Relationship between follicle size at insemination and pregnancy success. Proc Natl Acad Sci U S A 2005; 102:5268-73. Peters AR, Martinez TA, Cook AJ. A meta-analy sis of studies of the effect of GnRH 1114 days after insemination on pregnancy rates in cattle. Theriogenology 2000; 54:1317-26. Petersson KJ, Strandberg E, Gustafsson H, Berglund B. Environmental effects on progesterone profile measures of dairy co w fertility. Anim Reprod Sci 2005; 8: [in press]. Picton H, Briggs D, Gosden R. The molecu lar basis of oocyte development. Mol Cell Endocrinol 1998; 145:27–37. Plaizier JC, Lissemore KD, Kelton D, Ki ng GJ. Evaluation of overall reproductive performance of dairy herds. J Dairy Sci 1998; 81:1848-54. Pollard JW, Leibo SP. Comparative cryobiol ogy of in vitro and in vivo derived bovine embryos. Theriogenology 1993; 39:287. Portaluppi MA, Stevenson JS. Pregnancy rates in lactating dairy cows after presynchronization of estrous cycles and variations of the Ovsynch protocol. J Dairy Sci 2005; 88:914-21. Prather RS, First NL. Effect of cytochalas in B and demecolcine on freeze-thaw survival of murine embryos in vitr o. J Exp Zool 1986; 239:37-40. Pryce JE, Coffey MP, Simm G. The relati onship between body condition score and reproductive performance. J Dairy Sci 2001; 84:1508-15. Pursley JR, Mee MO, Wiltbank MC. Synchroni zation of ovulation in dairy cows using PGF2a and GnRH. Theriogenology 1995; 44:915.

PAGE 123

111 Pursley JR, Silcox RW, Wiltbank MC. Effect of time of artificial insemination on pregnancy rate, calving rates, pre gnancy loss, and gender ratio after synchronization of ovulation in lactati ng dairy cows. J Dairy Sci 1998; 81:2139-44. Pushpakumara PG, Gardner NH, Reynolds CK, Beever DE, Wathes DC. Relationships between transition period diet, metabolic pa rameters and fertility in lactating dairy cows. Theriogenology 2003;60:1165-85. Putney DJ, Drost M, Thatcher WW. Embr yonic development in superovulated dairy cattle exposed to elevated ambient temperature between days 1 to 7 post insemination. Theniogenology 1988a; 30:195-209. Putney DJ, Thatcher WW, Drost M, Wright JW, DeLorenzo MA. Influence of environmental temperature on reproductiv e performance of bovine embryo donors and recipients in the southwest region of the United States. Theriogenology 1988b; 30:905. Putney DJ, Malayer JR, Gross TS, Thatcher WW, Hansen PJ, Drost M. Heat stressinduced alterations in the s ynthesis and secretion of prot eins and prostaglandins by cultured bovine conceptuses and uter ine endometrium. Biol Reprod. 1988c; 39:717-28. Putney DJ, Mullins S, Thatcher WW, Drost M, Gross TS. Embryonic development in superovulated dairy cattle exposed to el evated ambient temperatures between the onset of estrus and insemination. Anim Reprod Sci 1989a; 19:37–51. Putney DJ, Drost M, Thatcher WW. Influen ce of summer heat stre ss on pregnancy rates of lactating dairy cattle following embr yo transfer or artificial insemination. Theriogenology 1989b; 31:765 78. Purcell SH, Beal WE, Gray KR. Effect of a CIDR insert and flunixin meglumine, administered at the time of embr yo transfer, on pregnancy rate and resynchronization of estrus in beef cattle. Theriogenology 2005; 64:867-78. Quintal-Franco JA, Kojima FN, Melvin EJ, Lindsey BR, Zanella E, Fike KE, Wehrman ME, Clopton DT, Kinder JE. Corpus lute um development and function in cattle with episodic release of luteinizing horm one pulses inhibited in the follicular and early luteal phases of the estrous cycle. Biol Reprod 1999; 61:921-6. Rabiee AR, Macmillan KL, Schwarzenberger F. Progesterone metabolism in ovariectomised non-lactating Holstein-Friesia n cows treated with progesterone with two levels of feed intake. Anim Reprod Sci 2001; 66:35-46. Rajamahendran R, Taylor C. Follicular dynamics and temporal relationships among body temperature, oestrus, the surge of luteinizing hormone and ovulation in Holstein heifers treated with norge stomet. J Reprod Fertil 1991; 92:461-67.

PAGE 124

112 Rajamahendran R, Sianangama PC. E ffect of human chorionic gonadotrophin on dominant follicles in cows: formation of accessory corpora lutea, progesterone production and pregnancy rates. J Reprod Fertil 1992; 95:577-84. Rakes AH, Owens MP, Britt JH, Whitlow LW. E ffects of adding beta-carotene to rations of lactating cows consuming different forages. J Dairy Sci 1985; 68:1732-7. Reichenbach HD, Liebrich J, Berg U, Brem G. Pregnancy rates and births after unilateral or bilateral transfer of bovine embryos produced in vitro. J Reprod Fertil 1992; 95:363-70. Rerat M, Zbinden Y, Saner R, Hammon H, Blum JW. In vitro embryo production: growth performance, feed efficiency, a nd hematological, metabolic, and endocrine status in calves. J Dairy Sci 2005; 88:2579-93. Rettmer I, Stevenson JS, Corah LR. Endoc rine responses and ovarian changes in inseminated dairy heifers afte r an injection of a GnRH agonist 11 to 13 days after estrus. J Anim Sci 1992a; 70:508-17. Rettmer I, Stevenson JS, Corah LR. Pregnanc y rates in beef cattle after administering a GnRH agonist 11 to 14 days after insemination. J Anim Sci 1992b; 70:7-12. Risco CA, Donovan GA, Hernandez J. Clinical mastitis associated with abortion in dairy cows. J Dairy Sci 1999; 82:1684–9. Rivera RM, Hansen PJ. Development of cultured bovine embryos after exposure to increased temperatures in the physiol ogical range. Reproduction 2001; 121:107-15. Rivlin J, Mendel J, Rubinstein S, Etkovitz N, Breitbart H. Role of hydrogen peroxide in sperm capacitation and acrosome r eaction. Biol Repr od 2004; 70:518-22. Rizos D, Fair T, Papadopoulos S, Boland MP, Lonergan P. Developmental, qualitative, and ultrastructural differences between ovine and bovine embryos produced in vivo or in vitro. Mol Reprod Dev. 2002; 62:320-7. Roberts JS, McCracken JA. Does PGF2 released from the uterus by oxvtocin mediate the oxvtocic action of oxytoc in? Biol Reprod 1976; 15:457-463. Robinson NA, Leslie KE, Walton JS. Eff ect of treatment with progesterone on pregnancy rate and plasma concentrations of progesterone in Holstein cows. J Dairy Sci 1989; 72:202-7. Robinson RS, Mann GE, Lamming GE, Wathes DC. Expression of oxytocin, oestrogen and progesterone receptors in uterine biopsy samples thr oughout the oestrous cycle and early pregnancy in cows. Reproduction 2001; 122:965-79.

PAGE 125

113 Rocha A, Randel RD, Broussard JR, Lim JM, Blair RM, Roussel JD, Godke R_A, Hansel W. High environmental temperature and humidity decrease oocyte quality in Bos taurus but not in Bos indicus cows. Theriogenology 1998; 49:657-65. Roche JF, Mackey D, Diskin MD. Reproductive management of postpartum cows. Anim Reprod Sci 2000; 60:703-12. Roman-Ponce H, Thatcher WW, Wilcox CJ. Hormonal interrelationships and physiological responses of l actating dairy cows to shade management system in a tropical environment. Th eriogenology 1981; 16:139–154. Ron M, Bar-Anan R, Wiggans GR. Factors affe cting conception rate of Israeli Holstein cattle. J Dairy Sci 1984; 67:854–60. Rose MT, Weekes TE, Rowlinson P. Individual variation in the milk yield response to bovine somatotropin in dairy cows. J Dairy Sci 2004;87:2024-31. Rosenberg M, Folman Y, Herz Z, Flamenbaum I, Berman A, Kaim M. Effect of climatic conditions on peripheral concentrations of LH, progesterone and oestradiol-17 beta in high milk-yielding cows. J Reprod Fertil 1982; 66:139-46. Roth Z. 1998. Immediate and delayed effect of heat stress on ovarian follicular development and function in dairy cows. MSc Thesis, Fac. Agric., Hebrew Univ., Rehovot, Israel, in Hebrew, with English abstract. Roth Z, Meidan R, Braw-Tal R, Wolfenson D. Immediate and delayed effects of heat stress on follicular development and its association with plas ma FSH and inhibin concentration in cows. J Reprod Fertil 2000; 120:83–90. Roth Z, Arav A, Bor A, Zeron Y, Braw-Tal R, Wolfenson D. Improvement of quality of oocytes collected in the autumn by enhan ced removal of impaired follicles from previously heat-s tressed cows. Reproduction 2001; 122:737–744. Roth Z, Hansen PJ. Involvement of apoptos is in disruption of developmental competence of bovine oocytes by heat shock duri ng maturation. Biol Reprod 2004; 71:1898906. Roth Z, Hansen PJ. Disruption of nuclear maturation and rearrangement of cytoskeletal elements in bovine oocytes exposed to heat shock during maturation. Reproduction 2005; 129:235-44. Rowson LE, Lawson RA, Moor RM. Production of twins in cattle by egg transfer. J Reprod Fertil 1971; 25:261-8. Royal M, Mann GE, Flint AP. Strategies for reversing the trend towards subfertility in dairy cattle. Vet J 2000; 160:53-60.

PAGE 126

114 Royal MD, Flint AP, Woolliams JA. Ge netic and phenotypic relationships among endocrine and traditional fertility traits a nd production traits in Holstein-Friesian dairy cows. J Dairy Sci 2002; 85:958-67. Rueda BR, Tilly KI, Hansen TR, Hoyer PB Tilly JL. Expression of superoxide dismutase, catalase and glutathione per oxidase in the bovine corpus luteum: evidence supporting a role for oxidative st ress in luteolysis. Endocrine 1995; 3:227–32. Ruoslahti E, Yamaguchi Y. Proteoglycans as modulators of growth factor activities. Cell 1991; 64:867-9. Rutledge, JJ. Use of embryo transfer and IVF to bypass effects of heat stress. Theriogenology 2001; 55:105-11. Ryan DP, Snijders S, Condon T, Grealy M, Sreenan J, O'Farrell KJ. Endocrine and ovarian responses and pregna ncy rates in dairy cows following the administration of a gonadotrophin releasing hormone analog at the time of artificial insemination or at mid-cycle post inseminati on. Anim Reprod Sci 1994; 34:179-91. Sangsritavong S, Combs DK, Sartori R, Arme ntano LE, Wiltbank MC. High feed intake increases liver blood flow and metabolism of progesterone and estradiol-17beta in dairy cattle. J Dair y Sci. 2002; 85:2831-42. Santos JE, Thatcher WW, Pool L, Over ton MW. Effect of human chorionic gonadotropin on luteal function and repr oductive performance of high-producing lactating Holstein dairy co ws. J Anim Sci 2001; 79:2881-94. Santos JEP, Cerri RLA, Ballou MA, Higginbotham GE, Kirk JH. Effect of timing of first clinical mastitis occurrence on lactational and reproductive performance on Holstein dairy cows. Anim Reprod Sci 2004a; 80:31–45. Santos JE, Juchem SO, Cerri RL, Galvao KN, Chebel RC, Thatcher WW, Dei CS, Bilby CR. Effect of bST and reproductive management on reproductive performance of Holstein dairy cows. J Dairy Sci 2004b; 87:868-81. Sartori R, Fricke PM, Ferreira JC, Ginther OJ, Wiltbank MC. Follicular deviation and acquisition of ovulatory capacity in bovine follicles. Biol Reprod 2001; 65:1403-9. Sartori R, Rosa GJM, Wiltbank MC. Ovaria n structures and circulating steroids in heifers and lactating cows in summer and lactating and dry cows in winter. J Dairy Sci 85; 2002:2813–22. Sartori R, Haughian JM, Shaver RD, Rosa GJ, Wiltbank MC. Comparison of ovarian function and circulating steroids in estrous cycles of Holstein heifers and lactating cows. J Dairy Sci 2004; 87:905-20.

PAGE 127

115 Savio JD, Boland MP, Roche JF. Developmen t of dominant follicles and length of ovarian cycles in post-partum dair y cows. J Reprod Fertil 1990; 88:581-91. Schmitt EJ, Diaz T, Drost M, Thatcher WW. Use of a gonadotropin-releasing hormone agonist or human chorionic gonadotropin for timed insemination in cattle. J Anim Sci 1996a; 74:1084-91. Schmitt EJ, Barros CM, Fields PA, Fields MJ, Diaz T, Kluge JM, Thatcher WW. A cellular and endocrine charac terization of the original and induced corpus luteum after administration of a gonadotropin -releasing hormone agonist or human chorionic gonadotropin on day five of the estrous cycle. J Anim Sci 1996b; 74:1915-29. Schneider B H, Sklan D, Chalupa W, Kronfeld DS. Feeding calcium salts of fatty acids to lactating cows. J Dairy Sci 1988; 71:2143–50. Schukken YH, Grommers FJ, Van de Geer D, Erb HN, Brand A. Risk factors for clinical mastitis in herds with a low bulk milk somatic cell count. 1. Data and risk factors for all cases. J Dairy Sci 1990; 73:3463-71. Scott TA, Shaver RD, Zepeda L, Yandell B, Smith TR. Effects of rumen-inert fat on lactation, reproduction, and health of high producing Holstein herds. J Dairy Sci 1995; 78:2435–51. Segerson EC Jr, Murray FA, Moxon AL, Redman DR, Conrad HR. Selenium/vitamin E: role in fertilization of bovine ova. J Dairy Sci 1977; 60:1001-5. Segerson EC, Ganapathy SN. Fertilization of ova in selenium/vitamin E-treated ewes maintained on two planes of nut rition. J Anim Sc i 1980; 51:386-94. Sheldon IM, Dobson H. Effects of gonadotr ophin releasing hormone administered 11 days after insemination on the pregnancy rates of cattle to the first and later services. Vet Rec 1993; 133:160-3. Sianangama PC, Rajamahendran R. E ffect of human chorionic gonadotropin administered at specific times follo wing breeding on milk progesterone and pregnancy in cows Ther iogenology 1992; 38:85-96. Shaw DW, Farin PW, Washburn SP, Britt JH Effect of retinol palmitate on superovulation rate and embryo quality in superovulated cattle.Theriogenology 1995; 44:51-8. Shiedlin A, Bigelow R, Christopher W, Ar abi S, Yang L, Maier RV, Wainwright N, Childs A, Miller R. Evaluation of hyaluronan from different sources: Streptococcus zooepidemicus, rooster comb, bovine vitreous, and human umbilical cord. Biomacromolecules 2004; 5:2122-27.

PAGE 128

116 Shilton KMF, Gayerie D, Hunter MG, Parkin son TJ, Lamming GE. Luteal inadequacy during the early luteal phase of sub-fe rtile cows. J. Repr od. Fertil 1990; 90:1–10. Silvia WJ, Taylor ML. Relationship betw een uterine secretion of prostaglandin F2 induced by oxytocin and endogenous concentr ations of estradiol and progesterone at three stages of the bovine estr ous cycle. J Anim Sci 1989; 67:2347-53. Sirois J, Fortune JE. Ovarian follicular dynamics during the estr ous cycle in heifers monitored by real-time ultras onography. Biol Reprod 1988; 39:308–17. Skarzynski DJ, Okuda K. Sensitivity of bovine corpora lutea to prostaglandin F2 is dependent on progesterone, oxytocin, a nd prostaglandins. Biol Reprod 1999; 60:1292-8. Sklan D, Bogin E, Avidar Y, Gur-arie S. Feeding calcium soaps of fatty acids to lactating cows: effect on production, body condition, and bl ood lipids. J. Dairy Res 1989; 56:675–81. Sklan D, Moallem U, Folman Y. Effect of feeding calcium soaps of fatty acids on production and reproductive responses in high producing lactating cows. J Dairy Sci 1991; 74:510–517. Sklan, D., M. Kaim, U. Moallem, and Y. Folm an. Effect of dietary calcium soaps on milk yield, body weight, reproductive hormones, and fertility in first parity and older cows. J Dairy Sci 1994; 77:1652–60. Smith JW, Legates JE. Relation of days ope n and days dry to lactation milk and fat yields. J Dairy Sci 1962; 45:1192-6. Spalding RW, Everett RW, Foote RH. Fertil ity in New York artificial inseminated Holstein herds in dairy herd im provement. J Dairy Sci 1975; 58:718-23. Son J, Grant RJ, Larson LL. Effects of tallow and escape protein on lactational and reproductive performance of dair y cows. J Dairy Sci 1996; 79:822–30. Soto P, Natzke RP, Hansen PJ. Identi fication of possible mediators of embryonic mortality caused by mastitis: Actions of lipopolysaccharide, prostaglandin F2 and the nitric oxide generator, sodium n itroprusside dihydrate, on oocyte maturation and embryonic development in cattle. Am J Reprod Immunol 2003; 50:263-72. Spencer TE, Bazer FW. Temporal and spatial alterations in uterine estrogen receptor and progesterone receptor gene expression during the estrous cycle and early pregnancy in the ewe. Biol Reprod 1995; 53:1527-43. Spencer TE, Ott TL, Bazer FW. -Interferon: pregnancy recogn ition signal in ruminants. Proc Soc Exp Biol Med 1996; 213:215–29.

PAGE 129

117 Spencer TE, Bazer FW. Biology of progest erone action during pregnancy recognition and maintenance of pregnanc y. Front Biosci 2002; 7:1879–98. Sreenan JM, Diskin MG. Early embryonic mort ality in the cow: its relationship with progesterone concentrati on. Vet Rec 1983; 112:517-21. Sreenan JM, Diskin MG. Effect of a unilateral or bilatera l twin embryo distribution on twinning and embryo survival rate in the cow. J Reprod Fertil 1989; 87:657-64. Short TH, Lawlor TJ, Everett RW. Inbreedi ng in US. Holsteins and its effect on yield and type traits. J Dairy Sci 1992; 75:154 [Abstr]. Stahl TJ, Conlin BJ, Seykora AJ, Steuerna gel GR. Characteristics of Minnesota dairy farms that significantly increased milk production from 1989-1993. J Dairy Sci 1999; 82:45-51. Staples CR, Thatcher WW, Clark JH. Relatio nship between ovarian activity and energy status during the early postpartum period of high producing dairy cows. J Dairy Sci 1990; 73:938-47. Staples CR, Burke JM, Thatcher WW. I nflu ence of supplemental fats on reproductive tissues and performance of lactat ing cows. J Dairy Sci 1998; 81:856-71. Stevenson JS, Schmidt MK, call EP. Factors a ffecting reproductive pe rformance of Dairy cows first inseminated after five we eks postpartum. J Dairy Sci 1983; 66:1148. Stevenson JS, Call EP, Scoby RK, Phatak AP. Double-insemination and gonadotropinreleasing hormone treatment of repeat-b reeding dairy cattle. J Dairy Sci 1990; 73:1766. Stevenson JS, Phatak AP, Rettmer I, Stewar t RE. Postinsemination administration of Receptal: Follicular dynamics, duration of cycle, hormonal responses, and pregnancy rates. J Dairy Sci 1993; 76:2536–47. Stojkovic M, Kolle S, Peinl S, Stojko vic P, Zakhartchenko V, Thompson JG, Wenigerkind H, Reichenbach HD, Sinowatz F, Wolf E. Effects of high concentrations of hyaluronan in culture medium on development and survival rates of fresh and frozen-thawed bovine embr yos produced in vitro. Reproduction 2002; 124:141-153. Stott GH, Wiersma F, Woods JM. Reproductive health program for cattle subjected to high environmental temperatures. J Am Vet Med Assoc. 1972 1; 161:1339-44. Stowe HD, Thomas JW, Johnson T, Marteni uk JV, Morrow DA, Ullrey DE. Responses of dairy cattle to long-term and short-term supplementation with oral selenium and vitamin E1. J Dairy Sci 1988;71:1830-9.

PAGE 130

118 Stronge AJ, Sreenan JM, Diskin MG, Mee JF Kenny DA, Morris DG. Post-insemination milk progesterone concentration a nd embryo survival in dairy cows. Theriogenology 2005; 64:1212-24. Taylor VJ, Cheng Z, Pushpakumara PG, Beever DE, Wathes DC. Relationships between the plasma concentrations of insulin-like growth factor-I in dairy cows and their fertility and milk yield. Vet Rec. 2004; 155:583-8. Thatcher WW, Chenault JR. Reproductive physiological respons es of cattle to exogenous prostaglandin F2 J Dairy Sci 1976; 59:1366-75. Thatcher WW, Collier RJ. Effects of clim ate on bovine reproduction. In: D. A. Morrow (Ed.) Current Therapy in Theriogenology 1986: 301 309. WB Saunders, Philadelphia. Thatcher WW, Macmillan KL, Hansen PJ, Dros t M. Concepts for regulation of corpus luteum function by the conceptus and ovari an follicles to improve fertility. Theriogenology 1989; 31:149-64. Thatcher WW, Driancourt MA, Terqui M, Badi nga L. Dynamics of ovarian follicular development in cattle following hysterect omy and during early pregnancy. Domest Anim Endocrinol 1991; 8:223-34. Thatcher WW, Staples CR, Danet-Desnoyers G, Oldick B, Schmitt EP. Embryo health and mortality in sheep and cattle. J Anim Sci 1994; 72:16. Thatcher WW, Meyer MD, Danet-Desnoyers G. Maternal recognition of pregnancy. J Reprod Fertil 1995; 49:15-28. Thatcher WW, De la Sota RL, Schmitt EJ, Di az TC, Badinga L, Simm en FA, Staples CR, Drost M. Control and management of ovarian follicles in cattle to optimize fertility. Reprod Fertil Dev 1996; 8:203-17. Thatcher WW, Binelli M, Burke JM, Staples CR, Ambrose JD, Coelho S. Antiluteolytic signals between conceptus and endome trium. Theriogenology 1997; 47:131–140 Thatcher WW, Mattos R, Moreira F, Binelli M, Ambrose JD. Experimental manipulation of follicular growth. Repr od Domest Anim 2000; 6:27–33. Thatcher WW, Moreira F, Santos JE, Matto s RC, Lopes FL, Pancarci SM, Risco CA. Effects of hormonal treatments on reproductive performance and embryo production. Theriogenology 2001; 55:75-89. Thatcher WW, Moreira F, Pancarci SM, Bartol ome JA, Santos JE. Strategies to optimize reproductive efficiency by regulation of ovarian function. Domest Anim Endocrinol 2002; 23:243-54.

PAGE 131

119 Thissen JP, Ketelslegers JM, Underwood LE. Nutritional regulation of the Insulin-like growth factors. Endocr Rev 1994; 15:80-101. Thomas MG, Williams GL. Metabolic hormone secretion and FSH-induced superovulatory responses of beef heifer s fed dietary fat supplements containing predominantly saturated and polyunsatur ated fatty acids. Theriogenology 1996; 45:451–8. Thompson JR, Everett RW, Hammerschmidt NL. Effects of inbreeding on production and survival in Holsteins. J Dairy Sci 2000a; 83:1856-64. Thompson JR, Everett RW, Wolfe CW. Effect s of inbreeding on production and survival in Jerseys. J Dairy Sci 2000b;83:2131-8. Tominaga K, Shimizu M, Ooyama S, Izai ke Y. Effect of lipid polarization by centrifugation at different developmental stag es on post-thaw survival of bovine in vitro produced 16-cell embryos. Theriogenology 2000; 53:1669-80. Toole BP, Zoltan-Jones A, Misra S, Ghatak S. Hyaluronan: a cr itical component of epithelial-mesenchymal and epithelial-carc inoma transitions. Ce lls Tiss Organs 2005; 179:66-72. Townson DH, Tsang PC, Butler WR, Frajblat M, Griel LC Jr, Johnson CJ, Milvae RA, Niksic GM, Pate JL. Relationship of fer tility to ovarian follicular waves before breeding in dairy cows. J Anim Sci 2002; 80:1053-8. Trout JP, McDowell LR, Hansen PJ. Character istics of the estrous cycle and antioxidant status of lactating Holstein cows expo sed to heat stress. J Dairy Sci 1998; 81:1244– 50. Twagiramungu H, Guilbault LA, Proulx J, Vill eneuve P, Dufour JJ. Influence of an agonist of gonadotropin-releasing hormone (Buserelin) on estrus synchronization and fertility in beef cows. J Anim Sci 1992; 70:1904-10. Ullah G, Fuquay JW, Keawkhong T, Clark BL Pogue DE, Murphey EJ. Effect of gonadotropin-releasing hormone at estrus on subsequent luteal function and fertility in lactating Holsteins during heat stress. J Dairy Sci 1996; 79:1950-3. Vajta G. Vitrification of the oocytes and em bryos of domestic animals. Anim Reprod Sci 2000; 60:357-64. Valcarcel A, de Matos DG, Furnus CC. The hyaluronic acid receptor (CD44) is expressed in bovine oocytes and preimp lantation stage embryos. Theriogenology 1999; 51:193 (Abstract). Van Cleef, J, Macmillan KL, Thatcher WW Lucy MC. Estrous synchronization and fertility in heifers treate d with CIDR before and after insemination. J Anim Sci 1989; 65:383 (Abstract).

PAGE 132

120 Van Wagtendonk-de Leeuw AM, Den Daas J HG, ThAM Kruip, Rall WF. Comparison of the efficacy of conventional slow fr eezing and rapid cryopreservation methods for bovine embryos. Cryobiology 1995; 32:157–67. Vasconcelos JL, Silcox RW, Rosa GJM, Pu rsley JR, Wiltbank MC. Synchronization rate, size of the ovulatory follicle, and pr egnancy rate after synchronization of ovulation beginning on different days of the estrous cycle in lactating dairy cows. Theriogenology 1999; 52:1067–78. Vasconcelos JL, Sartori R, Oliveira HN, Gu enther JG, Wiltbank MC. Reduction in size of the ovulatory follicle reduces subseque nt luteal size and pregnancy rate. Theriogenology 2001; 56:307-14. Vasconcelos JL, Sangsritavong S, Tsai SJ, Wiltbank MC. Acute reduction in serum progesterone concentrations after feed in take in dairy cows. Theriogenology 2003; 60:795-807. Vicini JL, Buonomo FC, Veenhuizen JJ, Miller MA, Clemmons DR, Collier RJ. Nutrient balance and stage of lactation affect re sponses of insulin, insulin-like growth factors I and II, and insulin-like growth factor-binding protei n 2 to somatotropin administration in dairy cows. J Nutr 1991;121:1656-64. Villa-Godoy A, Ireland JJ, Wortman JA, Ames NK, Hughes TL, Fogwel RL. Effect of ovarian follicles on luteal regression in heifers. J Anim Sci 1985; 60:519-27. Villarroel A, Martino A, BonDurant RH, Deletang F, Sischo WM. Effect of postinsemination supplementation with PRID on pregnancy in repeat-breeder Holstein cows. Theriogenology 2004; 61:1513-20. Viuff D, Greve T, Avery B, Hyttel P, Brockhoff PB, Thomsen PD. Chromosome aberrations in in vitro-produced bovine embryos at days 2-5 post-insemination. Biol Reprod 2000; 63:1143-48. Wall E, Brotherstone S, Kearney JF, Woollia ms JA, Coffey MP. Impact of nonadditive genetic effects in the estimation of breeding values for fertility and correlated traits. J Dairy Sci 2005; 88:376-85. Wang JY, Larson LL, Owen FG. Effect of beta-carotene supplementation on reproductive performance of dairy he ifers. Theriogenology 1982; 18:461-73. Wang X, Quinn PJ. The location and functi on of vitamin E in membranes. Mol Membr Biol 2000; 17:143-56. Wang X, Quinn PJ. Vitamin E and its func tion in membranes. Prog Lipid Res. 1999; 38:309-36. Webb R, Garnsworthy PC, Gong JG, Armstrong DG. Control of follicular growth: local interactions and nutritional influe nces. J Anim Sci 2004; 82:63-74.

PAGE 133

121 Wehrman ME, Welsh TH, Williams GL. Diet-i nduced hyperlipidemia in cattle modifies the intrafollicular cholesterol environmen t, modulates ovarian follicular dynamics, and hastens the onset of postpartum luteal activity. Biol Reprod 1991; 45:514–22. Westwood CT, Lean IJ, Garvin JK. Factors infl uencing fertility of Holstein dairy cows: a multivariate description. J Dairy Sci 2002; 85:3225-37. Wiggans GR, Ernst CA. Effect of genetic merit of sire for milk yield and herd yield level on reproductive traits. J Dairy Sci 1987; 70:232. Wiggans GR, VanRaden PM, Zuurbier J. Calc ulation and use of i nbreeding coefficients for genetic evaluation of United States dairy cattle. J Dairy Sci 1995; 78:1584-90. Willard S, Gandy S, Bowers S, Graves K, E lias A, Whisnant C. The effects of GnRH administration postinsemination on serum concentrations of progesterone and pregnancy rates in dairy cat tle exposed to mild summer heat stress. Theriogenology 2003; 59:1799-810. Wilson SJ, Marion RS, Spain JN, Spiers DE Keisler DH, Lucy MC. Effects of controlled heat stress on ovarian function of dairy cattle: 1. Lactating cows. J. Dairy Sci. 1998a 81:2124–31. Wilson SJ, Kirby CJ, Koenigsfeld AT, Keisler DH, Lucy MC. Effects of controlled heat stress on ovarian function of dairy ca ttle: 2. Heifers. J Dairy Sci 1998b; 81:2132– 38. Wilson RD, Weigel KA, Fricke PM, Rutledge JJ, Leibfried-Rutledge ML, Matthews DL, Schutzkus VR. In vitro production of Ho lstein embryos using sex-sorted sperm and oocytes from selected cull cows. J Dairy Sci 2005; 88:776-82. Wiltbank MC, Fricke PM, Sangsritavong S, Sartori R, Ginther OJ. Mechanisms that prevent and produce double ovulations in dairy cattle. J Dairy Sci 2000; 83:29983007. Wise ME, Armstrong DV, Huber JT, Hunter R. Wiersma F. Hormonal alterations in the lactating dairy cow in response to th ermal stress. J Dairy Sci 1988; 71:2480–85. Wolfenson D, Bartol FF, Badinga L, Barros CM, Marple DN, Cummings K, Wolfe D, Lucy MC, Spencer TE, Thatcher WW. Secretion of PGF2 and oxytocin during hyperthermia in cyclic and pregnant heifers. Theriogenology 1993; 39:1129 41. Wolfenson D, Thatcher WW, Savio JD, Badinga L, Lucy MC. The effect of a GnRH analogue on the dynamics of follicular deve lopment and synchronization of estrus in lactating dairy cows. Theriogenology 1994; 42:633-44. Wolfenson D, Thatcher WW, Badinga L, Sa vio JD, Meidan R, Lew BJ, Braw-Tal R, Berman A. Effect of heat stress on folli cular development during the estrous cycle in lactating dairy cattle Biol Reprod 1995; 52:1106–1113.

PAGE 134

122 Wolfenson D, Lew BJ, Thatcher WW, Graber Y, Meidan R. Seasonal and acute heat stress effects on steroid pr oduction by dominant follicles in cows. Anim Reprod Sci 1997; 47:9-19. Wolfenson D, Roth Z, Meidan R. Impaired reproduction in heat-stre ssed cattle: basic and applied aspects. Anim Re prod Sci 2000; 60-61: 535-47. Wolfenson D, Inbar G, Roth Z, Kaim M, Bl och A, Braw-Tal R. Follicular dynamics and concentrations of steroids and gonadotropi ns in lactating cows and nulliparous heifers. Theriogenology 2004; 15:1042-55. Wright S. Coefficients of inbreeding a nd relationship. Am. Natu ralist 1922; 56:330-338. Yasuda M, Nakano K, Yasumoto K, Tanaka Y. CD44: functional relevance to inflammation and malignancy. Hist ol Histopathol 2002; 17:945-50. Younas M, Fuquay JW, Smith AE ,Moore AB. Estrous and endocrine responses of lactating Holsteins to forced ventila tion during summer. J Dairy Sci 1993; 76:430– 6. Young C W, Seykora AJ. Estimates of inbr eeding and relationship among registered Holstein females in the United States. J Dairy Sci 1996; 79:502–5. Yoshioka K, Suzuki C, Arai S, Iwamura S, Hirose H. Gonadotropin-releasing hormone in third ventricular cerebrospinal fluid of the heifer during the estrous cycle. Biol Reprod 2001; 64:563–70. Zingg HH, Rozen F, Breton C, Larcher A, N eculcea J, Chu K, Russo C, Arslan A. Gonadal steroid regulation of oxytocin and oxytocin receptor gene expression. Adv Exp Med Biol 1995; 395:395-404. Zulu VC, Sawamukai Y, Nakada K, Kida K, Moriyoshi M. Rela tionship among insulinlike growth factor-I, blood metabolites a nd postpartum ovarian function in dairy cows. J Vet Med Sci. 2002a; 64:879-85. Zulu VC, Nakao T, Sawamukai Y. Insulinlike growth factor-I as a possible hormonal mediator of nutritional regulation of re production in cattle. J Vet Med Sci. 2002b; 64:657-65.

PAGE 135

123 BIOGRAPHICAL SKETCH C. Moiss Franco was born in 1979 in Sa nta Cruz, Bolivia. He is the youngest of three brothers (Inj. Oscar Antonio Franco; In j. Jorge Mauricio Franco) and three sisters (Dra. Rosario Franco; Mara Isabel Franco; Arq. Erika Lorena Franco). He is the son of Antonio V. Franco Monasterio (may god ble ss his soul) and Mercedes Yolanda Vaca ElHage. He graduated from La Salle High School in the same city in 1997 and enrolled the next year in the Department of Animal Science at the University of Arkansas in Fayetteville, USA, where he received his Bach elor of Science degree in animal science in 2001. During 2002 he did an internship in the Scottish Agricultural College with Dr Tom McEvoy. He enrolled in the graduate program of the Department of Animal Sciences at the University of Florida under supervision of Dr. Peter J. Hansen in January, 2003. He is currently a Master of Science candidate. Upon completion of his degree, he will open an embryo transfer company. In the near futu re he plans to resume his studies through pursuit of the Doctor of Philosophy degree at the University of Florida under Dr P.J. Hansen.


Permanent Link: http://ufdc.ufl.edu/UFE0013409/00001

Material Information

Title: Strategies to Enhance Fertility in Dairy Cattle during Summer including Use of Cryopreservation of in Vitro Produced Embryos
Physical Description: Mixed Material
Copyright Date: 2008

Record Information

Source Institution: University of Florida
Holding Location: University of Florida
Rights Management: All rights reserved by the source institution and holding location.
System ID: UFE0013409:00001

Permanent Link: http://ufdc.ufl.edu/UFE0013409/00001

Material Information

Title: Strategies to Enhance Fertility in Dairy Cattle during Summer including Use of Cryopreservation of in Vitro Produced Embryos
Physical Description: Mixed Material
Copyright Date: 2008

Record Information

Source Institution: University of Florida
Holding Location: University of Florida
Rights Management: All rights reserved by the source institution and holding location.
System ID: UFE0013409:00001


This item has the following downloads:


Full Text












STRATEGIES TO ENHANCE FERTILITY IN DAIRY CATTLE DURING SUMMER
INCLUDING USE OF CRYOPRESERVATION OF IN VITRO PRODUCED
EMBRYOS















By

C. MOISES FRANCO


A THESIS PRESENTED TO THE GRADUATE SCHOOL
OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT
OF THE REQUIREMENTS FOR THE DEGREE OF
MASTER OF SCIENCE

UNIVERSITY OF FLORIDA


2006


































Copyright 2006

by

C. Moises Franco





























This professional achievement reflects the sacrifice and guidance of my family especially
that of my mother, Mercedes Y. Vaca El-Hage, who laid the foundations with strong
pillars in my life.

This dissertation is dedicated to my beloved son Talyn Izaak Franco Benton and
astonishing father Antonio Vicente Franco Monasterio (Jf) for their endless love, support
and most important, inspiration.






"EL HOMBRE SE AUTORREALIZA EN LA MISMA MEDIA EN QUE SE
COMPROMETE AL CUMPLIMIENTO DEL SENTIDO DE SU VIDA"

Victor Frankl
(1905-1997)
















ACKNOWLEDGMENTS

This thesis would not have been possible without the enthusiasm, knowledge,

guidance, tenacity, and, perhaps most importantly faith that I received from my academic

advisor, Peter J. Hansen. From the very first interview to the last queries on research

accomplishments and career plans, he was always eager to entertain my ideas in hope that

I fulfilled my dreams) and become successful. I was not sure I could handle an

undertaking of such a magnitude, but was able to thanks to his consistent effort and true

desire to keep me on track.

I would like to extend my sincere appreciation to my committee member Dr. Karen

Moore, for her insight and willingness to help me academically without fail and regards

to time. Despite having other maj or responsibilities, Dr. Carlos Risco was willing to help

whenever asked. I thank him for his assistance and especially for the desire to help me

learn to palpate. Thanks are also extended to Dr. Alvin Warnick for his advice and

suggestions for improving my research proj ects and academic training. I would also like

to thank Dr. Joel Yelich for his teaching, support, and enthusiasm while providing me

with ideas that can help me achieve my goals.

Special thanks are extended to my family for encouraging me to seek for myself a

demanding and meaningful education. This thesis could not have taken place without that

precious gift.

Most sincere appreciation is also due to to my colleague and friend Dr. Rocio M.

Rivera, whose willingness to assist me in my early stages as a master's student helped to









kindle my interest in this exploration. I would not have gotten this far if it was not for her

unique and excellent training doing IVF. Dr. Zvi Roth was an inspirational friend whose

passion for science was transmitted to me. He also expressed his kindness and love

towards my son. I also thank Dr. Joel Hernandez for his support, friendship, and

guidance.

Thanks are given to Maria B. Padua for her assistance with the completion of this

manuscript and Luis Augusto Castro e Paula. Their unconditional friendship and help at

any given time is sincerely appreciated. I am grateful to Dean Jousan for making the

time to proofread my writings throughout the years and for his assistance in various

research experiments. Special thanks go to Amber Brad for her personality and joy that

helped the lab be united. Best of all has been my colleague and friend Jeremy Block for

his patience, expertise and engaging conversations that helped develop in me new

dreams. In addition, he always remained motivated throughout my transfer experiments.

I also would like to thank Central Packing Co. management and personnel at

Center Hill, FL, for providing the ovaries used for various experiments and William

Rembert for his assistance in collecting these ovaries. Special thanks go to Mary Russell

and Elise Griffin, for their assistance at the University of Florida Dairy Research Unit. I

thank Luther White and Mark Saulter of Hilltop Dairy, R.D. Skelton and Mathew Steed

of Levy County Dairy, and Mauricio Franco and Faby Grisel of Sausalito Dairy for

cooperation and assistance with the proj ects. And last but not least, I would like to thank

Todd Bilby, Osiloam Gomez, Reinaldo Cooke, Patrick Thompson, Saban Tekin, and

Paolette Soto.




















TABLE OF CONTENTS


IM Le

ACKNOWLEDGMENT S .............. .................... iv

LIST OF TABLES ............_ ..... ..__ ..............ix...


LI ST OF FIGURE S .............. ...............x.....

AB STRAC T ................ .............. xi


CHAPTER

1 REVIEW OF LITERATURE ..............._ ...............1.......... .....


Infertility in Modern Dairy Cattle............... .. .... ..............
Causes for the Decline in Fertility in Dairy Cattle .............. ...............2.....
Milk Yield .............. ........ ...............
Milk yield and energy balance .............. ...............3.....
Milk yield and endocrine milieu .............. ...............5.....
Milk yield and heat stress ................. ...............6............ ...
Milk yield and diseases ........................ ...............9
Milk yield, estrus detection, and fertility .............. ...............10....
Changes in Herd Size as a Factor in Reduced Fertility ................. ................. .11
Inbreeding ................... ...... .. ...... ........ ... ..... ..........1
Strategies to Improve Fertility in Lactating Dairy Cattle .................. ............... .....12
Treatment with Bovine Somatotropin (bST) to Enhance Fertility ......................13
Treatment with GnRH to Delay Luteolysis............. ... .................1
Increase in the Size of the Preovulatory Follicle to Generate a Larger Corpus
Luteum ............_.. ......_ ...... ............... 17.
Induction of an Accessory Corpus Luteum .........._.._ ....._.. ........._.._....19
Progesterone Supplementation .............. ...............20....
Inhibition of Luteolysis .............. ...............21....
Nutritional Strategies............... ........ .........2
Fat feeding to improve energy balance .............. ...............22....
Admini strati on of anti oxi dants ................. ...............25........... ..
Crossbreeding ................. ...............26.......... ......
Embryo Transfer............... ............ ... ... .......2
Limitations to Optimal Pregnancy Rates Using IVP TET .............. ..............28
Cryopreservation of IVP Embryos ...........__......_ ....__ ................30
Summary and Obj ectives of the Thesis ................. ...............31........... ..











2 EFFECTIVENESS OF ADMINISTRATION OF GONADOTROPIN
RELEASING HORMONE AT DAY 11, 14 OR 15 AFTER ANTICIPATED
OVTULATION FOR INCREASING FERTILITY OF LACTATING DAIRY
COWS AND NON-LACTATING HEIFERS .............. ...............34....

Introducti on ................. ...............34.................
M materials and M ethods ............... .... ......... ........................3
Experiment 1 GnRH Administration at Day 11 after Anticipated Ovulation
in Heifers Subj ected to Timed Artifieial Insemination during Heat Stress .....3 5
Experiment 2 GnRH Administration at Day 11 after Anticipated Ovulation
in Lactating Cows Subj ected to Timed Artifieial Insemination ....................37
Experiment 3 GnRH Administration at Day 14 after Anticipated Ovulation
in Lactating Cows Subj ected to Timed Artifieial Insemination ....................3 8
Experiment 4 GnRH Administration at Day 14 after Anticipated Ovulation
in Lactating Cows Subj ected to Timed Artifieial Insemination During Heat
Stress ................ ..... ..... ...... ...__ ..... ... .. .. .. ...........3
Experiment 5 GnRH Administration at Day 14 or Day 15 after Detected
Estrus............... ...............40
Statistical Analysis .............. ...............40....
R e sults................ ........... .... ..... ..__ ... ... .. .. ... ... .........4
Experiment 1 GnRH Administration at Day 11 after Anticipated Ovulation
in Heifers Subj ected to Timed Artifieial Insemination During Heat Stress ....42
Experiment 2 GnRH administration at Day 11 after Anticipated Ovulation
in Lactating Cows Subj ected to Timed Artifieial Insemination ....................42
Experiment 3 GnRH Administration at Day 14 after Anticipated Ovulation
in Lactating Cows Subj ected to Timed Artifieial Insemination ....................43
Experiment 4 GnRH Administration at Day 14 after Anticipated Ovulation
in Lactating Cows Subj ected to Timed Artifieial Insemination During Heat
Stress ................ ..... ..... ...... ... .. .. .. ................4
Experiment 5 GnRH Administration at Day 14 or Day 15 after Detected
E strus. ............... .... ............ .. ..... .. .............4
Overall Effectiveness of GnRH Treatment as Determined by Meta-Analysis....44
Discussion ........._..._. ...._ ... ...............44.....

3 EFFECT OF TRANSFER OF ONE OR TWO IN VITRO-PRODUCED
EMBRYOS AND POST-TRANSFER ADMINISTRATION OF
GONADOTROPIN RELEASING HORMONE ON PREGNANCY RATES OF
HEAT- STRES SED DAIRY CATTLE .....__.....___ ..........._ ...........5

Introducti on ........... __... ... ._ ...............52...
M materials and M ethods .............. ..... .............. ...... .......5
Experiment 1 Single or Twin Transfer of IVP Embryos into Crossbred
D airy R ecipients............ ..... .... ..........................5
Experiment 2 Administration of GnRH on Day 11 after Anticipated
Ovulation in Lactating Recipients that Received an IVP Embryo ..................57
Statistical Analysis .............. ...............59....
Re sults................. ...............60._ ___.......












Experiment 1 Single or twin transfer of IVP embryos ................. ................60
Pregnancy and calving rates ............... ....._ ....._ ............6
Characteristics of gestation, parturition, and calves.. .................. .................61
Experiment 2 Administration of GnRH on Day 11 after Anticipated
Ovul ati on ........._..... ...._... ...............62....
Discussion ........._..... ...._... ...............62.....


4 EFFECTS OF HYALURONIC ACID IN CULTURE AND CYTOCHALASIN B
TREATMENT BEFORE FREEZING ON SURVIVAL OF CRYOPRESERVED
BOVINE EMBRYO S PRODUCED IN VITRO ......____ ........ ...............72


Introducti on ............ .......__ ...............72...
M materials and M ethods .............. ...............73....

Embryo Production.................. .. .............7
Experimental Design and Embryo Manipulation ......____ ...... ...__...........74
Cryopreservation .............. ..... .. .............7
Thawing and Determination of Survival .............. ...............76....
Statistical Analysis .............. ...............76....
Re sults............ __.. ... ...._ ... ... .._ ... .......7
Effect of Hyaluronic Acid on Embryonic Development ..........._. ................... 77
Survival after Cryopreservation .............. ...............77....
Discussion ............ ..... .._ ...............78...


5 GENERAL DI SCU SSION ............_...... .__ ............... 2....


LIST OF REFERENCES ............_ .......__ ...............91...


BIOGRAPHICAL SKETCH ............_...... .__ ...............123...

















LIST OF TABLES


Table pg

2-1 Descriptive statistics, adjusted odds ratio (AOR) estimates, and 95% Wald
confidence intervals (CI) for effect of GnRH administration at Day 11 after
anticipated ovulation and ovulation synchronization protocol on pregnancy rates
of heifers during heat stress. ...._.._.._ ... ..._.__ ....___ ......._............49

2-2 Descriptive statistics, adjusted odds ratio (AOR) estimates, and 95% Wald
confidence intervals (CI) for effect of GnRH administration at Day 11 after
anticipated ovulation and season of insemination on pregnancy rates of lactating
cows subj ected to timed artificial insemination. ..........__......._ ..............50

2-3 Descriptive statistics, adjusted odds ratio (AOR) estimates, and 95% Wald
confidence intervals (CI) for effect of GnRH administration at Day 14 after
anticipated ovulation and season of insemination on pregnancy rates of lactating
cows subj ected to timed artificial insemination. ..........__......._ ..............50

2-4 Descriptive statistics, adjusted odds ratio (AOR) estimates, and 95% Wald
confidence intervals (CI) for effect of GnRH administration at Day 14 after
anticipated ovulation and Days in milk (<150 d vs > 150) at insemination on
pregnancy rates of lactating cows subj ected to timed artificial insemination
during heat stress. .........._ __..... ._ ...............51...

3-1 Effect of recipient type and number of embryos transferred per recipient on
pregnancy rates and losses. ............. ...............68.....

3-2 Effect of recipient type and number of embryos transferred per recipient on
characteristics of pregnancy and parturition. ................ .............. ........ .....69

3-3 Effect of recipient type and number of embryos transferred per recipient on
characteristics of calves born. ............. ...............70.....

4-1 Effect of hyaluronic acid added at day 5 after insemination on production of
blastocysts at day 7 and 8 after insemination. ............. ...............81.....

4-2 Effect of culture in hyaluronic acid and treatment with cytochalasin B on
survival after cryopreservation. ............. ...............81.....















LIST OF FIGURES


Finure pg

1-1 Rolling herd average (RHA, kg milk per lactation), calving interval (CI), and
services per conception (SPC) for 143 dairy herds continuously enrolled in the
Raleigh DHIA record system from 1970 to 1999. ............ .....................3

1-2 Temporal changes in first service pregnancy rate and annual average milk
production from high-producing Holstein-Friesian dairy herds in north-eastern
Spain. Data for pregnancy rate were recorded in the cool (October April
months) and warm season (May-September months). ........... ......................3

3-1 Maximum (open circles) and minimum (closed circles) daily air temperatures
and relative humidities (RH) during the experiments. .............. ....................7
















Abstract of Thesis Presented to the Graduate School
of the University of Florida in Partial Fulfillment of the
Requirements for the Degree of Master of Science

STRATEGIES TO ENHANCE FERTILITY IN DAIRY CATTLE DURING SUMMER
INCLUDING USE OF CRYOPRESERVATION OF IN VITRO PRODUCED
EMBRYOS


By

C. Moises Franco Vaca

May 2006

Chair: Peter J. Hansen
Major Department: Animal Sciences

There has been a precipitous decline in fertility of lactating dairy cows. In addition,

heat stress can further compromise fertility. The goals of this thesis were to 1) evaluate

strategies for enhancing fertility after artificial insemination using mid-cycle GnRH

treatment and 2) further develop embryo transfer using in vitro produced embryos as a

tool for increasing fertility. For the second obj ective, experiments tested whether

pregnancy rate could be improved by transfer of twin embryos and whether the

developmental competence of embryos after cryopreservation could be improved by

hyaluronic acid or cytochalasin B treatment.

A series of six experiments were conducted to test the efficacy of GnRH for

increasing fertility. Except for one experiment, in which GnRH administration at day 14

after insemination increased pregnancy rate, GnRH was without effect whether given at










day 11, 14 or 15 after insemination or at day 11 after anticipated ovulation in embryo

transfer recipients..

Neither unilateral transfer of two embryos nor administration of GnRH at Day 1 1

after anticipated ovulation improved pregnancy rates of dairy cattle exposed to heat

stress. Cytochalasin B treatment before freezing improved cryosurvival of bovine

embryos produced in vitro. In contrast, culture with hyaluronic acid was of minimal

benefit.

Taken together, GnRH treatment did not consistently increase pregnancy rates

when administered at Day 11-15 after insemination and is not recommended as a fertility-

enhancing treatment. Similarly, transfer of two embryos to the uterine horn ipsilateral to

the CL was not an effective method for increasing pregnancy rates in recipients. Transfer

of cryopreserved embryos may be enhanced by treatment of embryos with cytochalasin B

since this molecule increased in vitro survival, and it remains to be tested whether

survival of IVP embryos after vitrification can be improved by cytochalasin B treatment.















CHAPTER 1
REVIEW OF LITERATURE

Infertility in Modern Dairy Cattle

Fertility is defined as the ability of a cyclic animal to establish pregnancy and is an

important economic trait that affects herd productivity in dairy cattle (Pecsok et al., 1994;

Plaizier et al., 1998). Unfortunately, there has been a decline in fertility in dairy cows

over the last 10-40 years. Fertility, whether traditionally measured as conception rate

(number of pregnant animals divided by the number of inseminated animals) or herd

pregnancy rate (number of pregnant animals divided by the number of animals eligible to

be bred), has declined in North America (Butler, 1998), Ireland (Roche, 2000), Spain

(L6pez-Gatius, 2003), and the United Kingdom (Royal et al., 2000). Other important

reproductive measurements have changed during this time as well, including increases in

days to first service, days to conception, and calving interval (de Vries and Risco, 2005).

The magnitude of these changes in reproductive function over time is illustrated for data

from herds in the United States (Figure 1-1) and northeastern Spain (Figure 1-2).

The incidence of infertility of dairy cows has been correlated with changes in dairy

cattle physiology and improvements in genetic progress, nutrition, and management

practices. This literature review will seek to identify physiological causes for this

decrease in fertility and describe efforts to improve fertility.









Causes for the Decline in Fertility in Dairy Cattle

Milk Yield

The Animal Improvement Programs Laboratory of the United States Department of

Agriculture (USDA) has estimated the genetic trend for milk yield with an average of 37

kg/yr during the 1960s, 79 kg/yr during the 1970s, 102 kg/yr during the 1980s, and 116

kg/yr for the period from 1990 to 1996 (http://aipl.arsusda.gov; Hansen, 2000). It has

long been known that fertility is reduced in lactating cows as compared to non-lactating

heifers (Ron et al., 1984; Nebel and McGilliard, 1993). Given that milk yield has

increased over time as fertility has declined, the possibility must be considered that the

increase in milk yield is one reason that has contributed to the decreased fertility in dairy

cattle.

There are indications that the genetic correlation between female fertility and milk

production is antagonistic (Kadarmideen et al., 2000; Royal et al., 2002). In contrast,

Mahanna et al. (1979) suggested that there was no negative genetic correlation between

milk yield and reproduction because there was no difference in fertility among heifers

with different genetic abilities for milk yield. There may be an environmental effect of

milk yield on fertility, however. As described by Lucy (2001), the increase in milk yield

over the period from 1970 has been associated with a corresponding decrease in fertility

as measured by increased services per conception and calving interval (Figure 1-1).

According to Nebel and McGilliard (1993) there was little or no association of increased

milk yield compromising fertility prior to the 1970s (Gaines, 1927; Boyd et al., 1954;

Currie, 1956; Smith and Legates, 1962) but adverse effects of milk yield have been

correlated with reduced fertility in studies conducted since 1975 (Spalding et al., 1975;









Laben et al., 1982; Fonseca et al., 1983; Stevenson et al., 1983; Hillers et al., 1984;

Wiggans et al., 1987; Faust et al., 1988).

Using a data set of Holstein, Jersey, and Guernsey cows, it was found that 0.014

more services per conception were required for each additional 100 kg of 120-d milk for

Holsteins and 0.028 services per conception for Jersey and Guernsey cows (Olds et al.,

1979). Similarly, cows with the highest milk yield had the lowest first service conception

rate (Faust et al., 1988) or 90-d non-return rate (Al-Katanani et al., 1999) and highest

number of services (Faust et al., 1988). Days to first insemination and days open also

increased linearly as milk yield increased in Jersey dairy cattle (Fonseca et al., 1983).

Expression of estrus at first postpartum ovulation is less likely in cows with higher

milk production (Westwood et al., 2002). Some studies (Nielen et al., 1989; Kinsel et al.,

1998), but not others (Deluyker et al., 1991), correlate the incidence of twins to milk

yield. Amount of milk yield, however, was not correlated to increased incidence of

multiple ovulations (L6pez-Gatius et al., 2005b), yet the incidence of double ovulations

and twinning rate has increased in modern dairy cattle (Wiltbank et al., 2000). Taken

together, the associations of milk yield with reduced duration of estrus, increased days to

first insemination, increased number of inseminations per conception, reduced first

service conception rates, and reduced progesterone levels post-ovulation compromise

herd fertility.

Milk yield and energy balance

One way in which milk yield could affect fertility is through effects on energy

balance. A critical phase exists in the period following calving when dry matter intake

does not meet the increased metabolic demands of lactation, and as a result, the animal









enters a state classified as "negative energy balance" (NEB). During the period ofNEB,

body reserves of fat and protein are mobilized (Bauman and Currie, 1980; Butler and

Smith, 1989). An animal under NEB tends to have low body condition score (BCS), and

both NEB and low BCS are associated with low fertility (O'Callaghan, 1999; Butler,

2000; Pryce et al., 2001; Pushpakumara et al., 2003).

Energy deficiency reduces or impairs gonadotropin secretion, and as an animal

reaches this state around parturition, gonadotropin secretion to support follicular

development and ovulation is compromised and reproductive problems (i.e., cystic

ovaries) associated with onset of ovarian activity become prevalent (Zulu et al., 2002ab).

Growth hormone stimulates insulin-like growth factor 1 (IGF-1) production by the liver

(Jones and Clemmons, 1995), but during NEB growth hormone receptors are

downregulated in a process referred to as "Growth Hormone Resistance" (Donaghy and

Baxter, 1996). As milk production increases during early lactation and the cow is under

NEB, the liver becomes refractory to growth hormone because growth hormone receptors

are decreased (Vicini et al., 1991), and this result in reduced plasma concentration of

IGF-1 (Pell et al., 1993).

Follicular growth is stimulated by IGF-1 (Webb et al., 2004) and reduced plasma

concentrations of this growth factor are observed in cows with high milk yield (Rose et

al., 2004) and together are highly correlated to delayed return to ovarian cyclicity (Taylor

et al., 2004). After calving, cows with IGF-I concentrations greater than 50 ng/ml at first

service were 5 times more likely to conceive than those with lower concentrations

(Taylor et al., 2004).









The fact that high-producing cows have greater energetic demands for lactation

does not necessarily mean that these cows have greater NEB or low BCS. Staples et al.

(1990) found that low-producing cows had lower dry matter intake and were at a greater

risk for failure to conceive due to anestrus and infertility than high-producing cows. It

was observed that the low-producing group, classified as non-responders, sustained milk

production from 28% of body tissue reserve vs 15.9 and 16.7% in the early responder and

late responder groups. This interaction was confirmed when low-producing cows had

lost the most body weight during the first 2 weeks of lactation and were in the greatest

energy deficit (Staples et al., 1990).

Milk yield and endocrine milieu

Cows displaying greater milk production often have higher dry matter intakes

(Staples et al., 1990; Hommeida et al., 2004), which has been demonstrated to decrease

circulating progesterone concentrations in lactating (Hommeida et al., 2004) and non-

lactating cows (Rabiee et al., 2001). Acute feeding reduced circulating progesterone by

25% in pregnant cows (Vasconcelos et al., 2003). Lucy and co-workers (1998) found

that circulating progesterone was lower in cattle genetically selected for high milk

production.

Sangsritavong et al. (2002) demonstrated that lactating cows have a much greater

steroid metabolism than non-lactating cows. As a result, lactating cows may have larger

luteal tissue volume on the ovary (Sartori et al., 2002; Sartori et al., 2004) yet experience

lower circulating progesterone and estradiol concentrations than heifers and dry cows (De

la Sota et al., 1993; Wolfenson et al., 2004). There is evidence that low progesterone









secretion can compromise fertility in dairy cattle (Mann and Lamming, 1999) and an

increase in progesterone secretion may facilitate embryonic development.

Progesterone provides nourishment for the concepts via induction of secretion of

proteins and other molecules from the endometrium (Garrett et al., 1988a). Low

peripheral concentrations of progesterone are also associated with increased luteinizing

hormone (LH) pulses (Ireland and Roche, 1982) that can stimulate luteolytic signals in

favor of pregnancy failure. Skarzynski and Okuda (1999) reported that blocking the

progesterone receptor with a progesterone antagonist (onapristone) increased

prostaglandin F2a (PGF2a) prOduction by bovine luteal cells harvested from mid-cycle

corpora lutea (CL) (Days 8-12). In addition, it was revealed that the bovine corpus

luteum (CL) does not undergo apoptosis until progesterone production has declined

(Juengel et al., 1993; Rueda et al., 1995).

Milk yield and heat stress

One reason why milk yield might decrease fertility of lactating cows is because it

increases their susceptibility to heat stress. Infertility is a particular problem during heat

stress (Ingraham et al., 1974; Putney et al., 1989b; Al-Katanani et al., 1999) and air

temperatures as low as 27oC can induce hyperthemia in lactating dairy cows (Berman et

al., 1985). Cows exposed to elevated temperatures to induce heat stress experienced

reduced pregnancy rates (Dunlap and Vincent, 1971) and increased embryonic mortality

(Putney et al., 1988ab; Ealy et al., 1993). On the other hand, provision of cooling in the

summer increased pregnancy rates as compared to non-cooled cows (Stott et al., 1972;

Roman-Ponce et al., 1981; Ealy et al., 1994).









The ability to regulate body temperature during heat stress is exacerbated by

lactation because of the excess heat production. The increase in body temperature in

response to heat stress is greater for lactating cows than heifers (Cole and Hansen, 1993)

and greater for high-producing cows than low-producing cows (Berman et al., 1985).

Data collected on fertility at first service from 8124 Holstein cows located in South

Georgia as well as North and South Florida support the idea that a high level of milk

production reduces fertility of lactating cows. When cows were grouped according to

mature equivalent milk yield, there was a milk yield class x month of breeding interaction

that resulted from the fact that the duration and magnitude of summer infertility increased

as milk yield increased (Al-Katanani et al., 1999).

Heat stress before, shortly after, and on the day of breeding is associated with

reduced fertility. Heat stress can compromise fertility throughout various reproductive

processes such as oocyte developmental competence (Picton et al., 1998; McNatty et al.,

1999) since the oocyte becomes sensitive to damage throughout the various stages of

follicular growth (Badinga et al., 1993). Indeed, follicular steroidogenesis, follicular

dynamics and altered concentrations of FSH and inhibin become altered in response to

heat stress (Badinga et al., 1994; Wolfenson et al., 1997; Roth et al., 2000). During heat

stress sperm can be damaged after insemination due to the generation of reactive oxygen

species (Ishii et al., 2005) and embryonic development can be compromised directly

(Monty et al., 1987). Not surprisingly the heat stress problem is multifactorial (Hansen et

al., 2001).

Heat stress of superovulated cows at day 1 after breeding reduced the proportion of

embryos that were blastocysts at day 8 after breeding, but heat stress on day 3, 5 or 7









after breeding did not affect subsequent embryonic development (Ealy et al., 1993).

Superovulated heifers experienced a high percentage of retarded embryos recovered on

day 7 after insemination after exposure to high temperature and humidity at the onset of

estrus for 10 h (Putney et al., 1989a). In another study heat stress was induced in

Holstein heifers by submitting them from day 1 to day 7 after estrus to 42oC for 7 h

(treatment) or 30oC for 16 h (control) and results obtained revealed more retarded

embryos with degenerate blastomeres on the day of recovery (20.7% vs. 51.5%,

respectively; Putney et al., 1988a).

One cause for the observed reduction in reproductive performance under heat stress

conditions is steroidogenic capacity and its effects on oocyte function (Roth et al., 2001;

Al-Katanani et al, 2002b; Roth and Hansen, 2004). Under heat stress, low estradiol

concentration in the follicular fluid of dominant follicles involves reduced aromatase

activity in the granulosa cells (Badinga et al., 1993) and reduced androstenedione

production by theca cells (Wolfenson et al., 1997). Although earlier studies were

inconsistent in demonstrating that plasma concentrations of estradiol are reduced under

heat stress (no change- Gwazdauskas et al., 1981; increase Rosenberg et al., 1982;

decrease Gwazdauskas et al., 1981), recent work points toward heat stress resulting in

lower estradiol concentrations in the follicular fluid (Badinga et al., 1993; Wolfenson et

al., 1995; Roth, 1998; Wilson et al., 1998ab).

Heat stress also has been reported to decrease (Rosenberg et al., 1982, Younas et

al., 1993; Howell et al., 1994), increase (Abilay et al., 1975; Roman-Ponce et al., 1981;

Trout et al., 1998), or have no effect (Wise et al., 1988; Wolfenson et al., 1995) on

peripheral concentrations of progesterone. Elevated temperatures in culture can directly









influence endometrium explants by increasing PGF2a Secretion (Putney et al., 1988c;

Malayer and Hansen, 1990) and from days 8-16 of pregnancy can reduce the size of the

embryo at day 17 (Biggers et al., 1987).

A retrospective survey involving 12,711 lactations from high-yielding dairy herds

in northeast Spain demonstrated that milk yield per cow increased from 1991-2000

(L6pez-Gatius, 2003; see Figure 2). For each 1000 kg increase in average milk yield in

the warm period, there was a decrease of 6% in pregnancy rate, and 7.6% in cyclicity,

and an increase of 8% in the incidence of inactive ovaries. During the cool period,

however, there was no change in fertility over time. Thus, the continual increase in milk

yield might have reduced fertility in Spain, at least, by exacerbating effects of heat stress.

Milk yield and diseases

Increased incidence of certain diseases has been associated with elevated milk

yield. High somatic cell score and clinical mastitis (Schukken et al., 1990; Barkema et

al., 1998; Chassagne et al., 1998; Fleischer et al., 2001); lameness (Green et al., 2002);

cystic ovarian disease (Fleischer et al., 2001; L6pez-Gatius et al., 2002); milk fever

(Fleischer et al., 2001); and acute metritis (Kelton et al., 1998) are all correlated with

milk yield.

Compared to non-mastitic herd-mates, high producing cows were at a greater risk

of developing clinical mastitis (Grahn et al., 2004). Number of days to conception,

artificial inseminations per conception and number of days to first artificial insemination

(AI) were significantly greater for cows with clinical mastitis (Barker et al., 1998), and

may affect embryonic survival when occurring after insemination (Soto et al., 2003).

According to Jousan et al., (2005) an elevated somatic cell count score among lactating









females influenced mid-to-late fetal loss (represented as occurring after day 70 to 90 of

gestation) and mastitis has been reported to affect pregnancy loss during the period of

embryonic (Chebel et al., 2004) and fetal development (Risco et al., 1999; Santos et al.,

2004a).

High yielding cows had an increased likelihood of becoming lame (Green et al.,

2002) and cows that had been treated for lameness had a negative influence on pregnancy

to Birst insemination and numbers of inseminations per service period (Petersson et al.,

2005). Similarly, non-lame cows were more likely to conceive at first service than lame

cows and lameness within the first 30 days after calving was associated with reduced

pregnancy rates at first AI and a higher number of services per conception (Hernandez et

al., 2001; Melendez et al., 2003). In a meta-analysis of several published papers, leg

problems were associated with an average increase of 12 days to conception (Fourichon

et al., 2000).

Cows that develop cysts remain infertile as long as this condition persists and early

spontaneous cyst recovery was negatively correlated with milk yield (L6pez-Gatius et al.,

2002). Similarly, elevated milk yield increased the risk of cows developing cysts (L6pez-

Gatius et al., 2002) and days from metritis occurrence to first AI is also correlated to

infertility (Loeffler et al., 1999). Milk yield in the current lactation is also correlated with

incidence of milk fever (Fleischer et al., 2001) and this disease reduces fertility (Chebel

et al., 2004).

Milk yield, estrus detection, and fertility

Milk yield may affect fertility indirectly by reducing the ability to accurately detect

estrus. An antagonistic relationship between increased milk production and days to first









visual estrus has already been reported. According to LC~pez et al. (2004), duration,

standing events, intensity (determined by the number of standing events per hour), and

standing time were reduced for high-producing cows as compared to low producers.

Similarly, Harrison et al. (1990) reported that elevated milk yield was correlated to a

longer period of estrus suppression. Westwood et al., (2002) indicated that high genetic

merit for milk yield influenced significantly the chance a cow showed weak signs of

estrus as compared to low milk producing cows.

Cows with elevated milk yield also had reduced circulating estradiol concentrations

on the day of estrus expression and shorter duration of estrus despite having larger

preovulatory follicle diameters (LC~pez et al., 2004).

Changes in Herd Size as a Factor in Reduced Fertility

Increased milk yield is not the only change in dairy farming over the last 50 years

and some of these other changes could also contribute to decreased fertility. One major

change has been the trend towards large farms. In a review, Lucy et al. (2001) cited data

from the USDA National Agricultural Statistics Services that nearly 30% of all dairy

farms in the United States have more than 500 cows. In addition, Stahl et al. (1999)

reported that the expansion of dairy herds comes in large part through the purchase of

first-lactation cows. Thus, as Lucy et al. (2001) pointed out, these more infertile

primiparous cows (Stahl et al., 1999) may have represented an increasingly larger

percentage of the herd as dairy herds have expanded over the last 10-40 years. The

importance of changes in herd size as a cause for infertility have been questioned by de

Vries and Risco (2005) who found no clear association with reproductive function.

Nevertheless, as the herd size is increased one would expect that the likelihood that

it becomes harder for accurately detecting estrus becomes a challenge because factors









associated with herd size such as the surface (concrete floor) on which the cow stands

will reduce the preponderance of cows displaying estrus activity (Britt et al., 1986;

O'Connor and Senger, 1997).

Inbreeding

Inbreeding represents increased frequency of identical alleles at a gene locus and

the inbreeding percent is a measure for the genes of an individual that are identical by

descent (Wright, 1922; Falconer, 1981). It is generally considered that reproductive

function declines when inbreeding levels in a population rise above 6.25% (Hansen et al.,

2005). Increased degree of inbreeding as the result of use of AI could explain some of

the declines in fertility experienced by dairy cattle because inbreeding coefficients have

increased in all the maj or U. S. dairy breeds. Estimates of inbreeding in the U. S. dairy

population are near 5% currently (Short et al., 1992; Wiggans et al., 1995; Young et al.,

1996; Hansen, 2000; Wall et al., 2005) and increasing at a constant rate of about 0. 1% per

year for U. S. Holsteins (Hansen et al., 2005). At an average of 5%, it is likely that many

dairy cows have inbreeding coefficients above 6.25% (Hansen et al., 2005).

Thompson et al. (2000ab) found calving intervals to increase by 12 and 17 d for

Jersey and Holsteins cows, respectively, with levels of inbreeding >10%. Similarly,

inbreeding had pronounced negative effects on fertility at higher levels (10%) of

inbreeding (Wall et al., 2005). In another study, animals with an inbreeding coefficient

>9% had fewer transferable embryos following superovulation than animals with a lower

inbreeding coefficient (Alvarez et al., 2005).

Strategies to Improve Fertility in Lactating Dairy Cattle

Four general approaches to improve reproductive function in dairy cattle have

been developed. The first is to regulate the timing of ovulation using gonadotropin









releasing hormone (GnRH) and PGF2 O utilized in timed AI (TAI) programs. The

advantage of this approach is that this program maximizes the number of animals

inseminated and allows inseminations to be made at some pre-planned time to eliminate

the need for estrus detection. Pioneering studies (Thatcher et al., 1989; Twagiramungu et

al., 1992; Wolfenson et al., 1994) were able to synchronize estrus effectively, however,

subsequent studies at the University of Florida (Schmitt et al., 1996a) and University of

Wisconsin (Pursley et al., 1995) led to the development of the Ovsynch TAI program and

the demonstration that good pregnancy rates can be achieved (Thatcher et al., 2001;

Thatcher et al., 2002). Although this approach is an effective one and is widely used in

dairy herds, it involves regulation of events occurring before conception and is beyond

the scope of the present review. The second approach is to use information regarding the

hormonal basis for establishment of pregnancy and signaling between the maternal and

embryonic units during early pregnancy as the basis for pharmacological treatments to

improve embryonic survival. Failure of essential biochemical dialogue between the

concepts and the maternal unit undoubtedly contributes to embryonic mortality and

termination of pregnancy (Spencer et al., 1996; Spencer and Bazer, 2002). The third

approach has been to regulate the nutrition of the dairy cow to improve energy balance or

to provide specific nutrients that favor establishment and maintenance of pregnancy.

Finally, recent work has focused on use of embryo transfer to bypass early embryonic

death and perhaps coupled with crossbreeding may become an important alternative since

Holsteins have become more inbred (Hansen et al., 2005).

Treatment with Bovine Somatotropin (bST) to Enhance Fertility

Circulating concentrations of IGF-I, glucose, and cholesterol are reduced in

lactating animals (de la Sota et al., 1993; Beam and Butler 1997). Circulating









concentrations of IGF-I is influenced by nutrition (Adam et al., 1997) and closely related

to energy balance of the cow (Ginger et al., 1997; Beam and Butler, 1998; 1999). Present

in serum and in various tissues, IGF-I is produced mainly by the liver but other organs as

well (Murphy et al., 1987; Thissen et al., 1994). IGF-I regulates ovarian function in dairy

cattle (Breukink et al., 1998; Chase et al., 1998), is necessary for proper follicular

development in which a fully competent oocyte capable of inducing ovulation develops

(Lucy et al., 1992a), and is required for normal CL formation and function (Leeuwenberg

et al., 1996; Chase et al., 1998). Dairy cows that initiated estrous cyclicity during the

postpartum period had higher plasma IGF-I than anestrous cows (Thatcher et al., 1996),

cystic and inactive ovary or persistent CL cows (Zulu et al., 2002a).

Bovine somatotropin (bST) increases plasma concentrations of insulin, IGF-I, and

growth hormone (Bilby et al., 2004), perhaps by stimulating ovarian function especially

after IGF-1 plasma levels are reduced in lactating animals (de la Sota et al., 1993). In

addition, inj section of bST stimulates concepts growth by day 17 of pregnancy (Bilby et

al., 2004). Additional studies provided evidence that bST can improve pregnancy rates in

lactating cows (Moreira et al., 2000b; Morales-Roura et al., 2001; Santos et al., 2004b).

Superovulated donor cows that received bST treatment experienced reduced number of

unfertilized oocytes, increased number of embryos that developed to the blastocyst stage,

and increased number of transferable embryos (Moreira et al., 2002). Collectively, these

studies indicate that critical thresholds of GH and IGF-I concentrations are needed to

stimulate reproductive performance (Bilby et al., 2004).

Treatment with GnRH to Delay Luteolysis

The estrous cycle is characterized by 2, 3, and sometimes 4 waves of follicular

growth (Sirois and Fortune, 1988; Ginther et al., 1996). During the second half of the









luteal phase, development of an estrogenic follicle facilitates the luteolytic process via

secretion of estradiol. Non-pregnant cows have higher peripheral concentrations of

estradiol on days 16 and 18 after breeding compared to pregnant animals (Ahmad et al.,

1997). Thatcher et al. (1991) examined the largest and second largest follicles present on

day 17 after estrus in pregnant and cyclic dairy cows. In the cyclic cows, the largest

follicle had greater aromatase activity and contained more estradiol and less progesterone

in the follicular fluid than the second largest follicle. These relationships were reversed

in pregnant animals, which indicated an earlier recruitment of the third wave of follicular

development in the pregnant animal associated with delayed luteolysis and higher

pregnancy rates. That these follicles play an important role in luteolysis was shown by

Villa-Godey et al. (1985), who reported that electrocautery to destroy large follicles was

associated with an extension of the estrous cycle.

Estradiol is now known to be one of three hormones that control uterine secretion

of PGF2,, With progesterone and oxytocin also being involved. Pulsatile release of PGF2,

from the luminal epithelium of the endometrium is stimulated via oxytocin (Roberts and

McCracken, 1976; Silvia and Taylor, 1989; Milvae and Hansel, 1980). Progesterone and

estradiol regulate this process because estradiol induces formation of oxytocin receptors

(Silvia and Taylor, 1989; Zingg et al., 1995; Robinson et al., 2001) after progesterone

exposure (Ginther, 1970; Garrett et al., 1988b; Lafrance and Goff, 1988). While

progesterone initially suppresses PGF2, Secretion by blocking oxytocin receptors during

the early and mid-luteal phase of the estrous cycle, the endometrium becomes responsive

to oxytocin and progesterone receptors become down regulated as the estrous cycle

progresses (Lafrance and Goff, 1988; Spencer and Bazer, 1995).










Delaying luteolysis might improve pregnancy rate by allowing embryos more time

to produce sufficient quantities of interferon-z (IFN- z). Eliminating or decreasing

estradiol production from the dominant follicle during the critical period of early

pregnancy could be one strategy to improve pregnancy establishment (Thatcher et al.,

2000; Binelli et al., 2001). One approach for doing this is to use GnRH to regulate

follicular function.

Gonadotropin releasing hormone is a decapeptide that plays a central role in

regulating reproductive processes. Release of GnRH from the hypothalamus occurs in a

pulsatile fashion and can be regulated by various internal and external signals.

Hypothalamic GnRH is synthesized in cell bodies of neurosecretory neurons, and is

transported to and released from the median eminence into the hypothalamic-

hypophyseal portal system (Loucopoulos and Ferin, 1984). GnRH has its primary effects

at the pituitary gonadotrope and stimulates the pulsatile release of the gonadotropins

luteinizing hormone (LH) and follicle-stimulating hormone (FSH) into the peripheral

circulation (Chenault et al., 1990). Two potential gonadotropin responsive tissues within

the ovary are the CL and the follicle. LH release induces ovulation or luteinization of

large ovarian follicles present at the time of treatment (Thatcher and Chenault, 1976).

One strategy tested for increasing pregnancy rate is to inject GnRH or GnRH

analogues at day 11-14 after estrus to increase progesterone secretion (Willard et al.,

2003) and delay luteolysis (Macmillan and Thatcher, 1991), thereby increasing the

chance for an embryo to initiate its own antiluteolytic mechanism. Inj section of GnRH at

this time can lead to decreased estrogen secretion (Rettmer et al., 1992a; Mann and









Lamming, 1995a) in an action that likely involves luteinization of the dominant follicle

(Thatcher et al., 1989; Rettmer et al., 1992a; Ryan et al., 1994).

Improvement of fertility has been seen by administration of GnRH or its analogues

at day 1 1-14 in nulliparous beef heifers (Rettmer et al., 1992b) and lactating dairy cows

(Macmillan et al., 1986; Lajili et al., 1991; Sheldon and Dobson, 1993; Drew and Peters,

1994; Willard et al., 2003; L6pez-Gatius et al., 2005a). In contrast to these positive

results, there was no favorable effect of similar treatments of GnRH or GnRH analogues

on pregnancy rates in other studies (Jubb et al., 1990; Stevenson et al., 1993; Ryan et al.,

1994; Bartolome et al., 2005). In a meta-analysis of published results, Peters et al. (2000)

concluded that the overall effect of GnRH administration between day 11 and 14 after

anticipated ovulation was positive but that results were not consistent between studies.

Increase in the Size of the Preovulatory Follicle to Generate a Larger Corpus
Luteum

As mentioned earlier, high-yielding dairy cows are more likely to have lower

circulating concentrations of progesterone throughout the estrous cycle than cows with

lower milk yields because of increased rate of progesterone catabolism (Lucy et al., 1998;

Vasconcelos et al., 1999). Given the importance of progesterone concentration for

embryonic survival (Man and Lamming, 2001), efforts have been made to increase

progesterone secretion in cows. One possible effect of mid-cycle treatment with GnRH is

to increase progesterone secretion (Schmitt et al., 1996b; Willard et al., 2003). Another

approach for increasing progesterone concentrations has been to regulate the size of the

preovulatory follicle to affect subsequent CL function.

Optimum differentiation and growth rate of the CL varies according to the duration

and amplitude of the ovulatory LH surge such that inhibition of LH release preceding the










preovulatory surge of LH resulted in development of a smaller CL in diameter (Quintal-

Franco et al., 1999). Induced ovulation of small follicles resulted in a smaller CL and

reduced secretion of progesterone than when a larger follicle ovulated (Vasconcelos et

al., 2001). In another study (Perry et al, 2005), regression analysis indicated that

pregnancy rate for cows with induced ovulation with an ovulating follicle of 14.5 mm

was higher than for cows ovulating follicles <10.3 mm in diameter. It was further

revealed that 39% of cows that lost their pregnancy had ovulatory follicles <11 mm in

diameter. Among cows that ovulated spontaneously, however, pregnancy rates at day 27

and 68 were independent of ovulatory follicle size (Perry et al., 2005). In contrast to this

result, Vasconcelos et al (1999) found that the group of cows ovulating larger follicles

had lower pregnancy rates on day 28 and 98 after AI and higher pregnancy loss between

these times.

Administration of GnRH just prior to or at the time of the LH surge causes an

amplified preovulatory surge ofLH (Lucy and Stevenson, 1986; Yoshioka et al., 2001).

Inj section of GnRH at or near the time of estrus increased the proportion of large luteal

cells in the CL on day 10 of the estrous cycle (Mee et al., 1993), peripheral progesterone

concentrations during the first 7 days of the estrous cycle (Lucy and Stevenson, 1986),

and increased pregnancy rates in repeat breeding cows (Stevenson et al., 1990; Mee et al.,

1993).

Ullah et al. (1996) observed that GnRH treatment at estrus in dairy cows improved

pregnancy rates and increased peripheral progesterone concentration. Conversely, GnRH

administered to lactating dairy cows at the time of AI did not affect pregnancy rates

(Ryan et al., 1994). Similarly, Mee et al. (1990) concluded that GnRH treatment at 1 h or









12 to 16 h after first detected estrus did not improve pregnancy rates at first service. Mee

et al. (1990) mentioned that 16 studies in the literature suggest an overall advantage in

pregnancy rate of 6 percentage points (53 vs. 59%) or an 1 1% improvement for cows

receiving GnRH treatment at the time of AI or up to 6 h preceding AI.

Induction of an Accessory Corpus Luteum

Progesterone concentrations following ovulation have been positively correlated to

volume of uterine secretions (Garrett et al., 1988a), concepts development (Garrett et

al., 1988a; Mann et al., 1996), the embryos ability to secrete IFN-z (Kerbler et al., 1997;

Mann et al., 1998), embryo viability for subsequent survival (Stronge et al., 2005), and

perhaps most importantly conception rates (Hansel, 1981; Fonseca et al., 1983; Shilton et

al., 1990; Larson et al., 1997). One possible approach to increasing progesterone

secretion has been to induce formation of an accessory CL by administering GnRH or

hCG, LH or their analogues at a time when the first wave dominant follicle is present

after ovulation (metestrus) (Rajamahendran and Sianangama, 1992; Schmitt et al., 1996b;

Santos et al., 2001). Santos et al. (2001) reported that hCG treatment on d 5 of a

synchronized estrous cycle induced an accessory CL in 86.2% of treated cows, increased

plasma progesterone by 5 ng/ml, and increased conception rates on day 28 from 38.7% to

45.8% and on day 90 of pregnancy from 3 1.9% to 38.4%. Lactating dairy cows treated

with GnRH on d 5 (Willard et al., 2003) and hCG on day 7 (Raj amahendran and

Sianangama, 1992) or day 4 in heifers (Breuel et al., 1989) reported successful accessory

CL formation and an increase in conception rates and pregnancy rate.

Besides stimulating luteal tissue formation, treatment of cows to induce ovulation

of the first wave dominant follicle with GnRH or GnRH analogues also reprograms

follicular growth to increase the proportion of estrous cycles composed of three follicular









waves as compared to two waves (Diaz et al., 1998). Such an effect could reduce the

probability that a large, highly estrogenic follicle is present during the critical period of

pregnancy recognition. Compared to animals with two-wave cycles, Holstein cows

(Townson et al., 2002) and beef cows (Ahmad et al., 1997) with a three-wave cycle had

higher conception rates and a longer luteal phase (Ginther et al., 1989).

Progesterone Supplementation

The ability of the concepts to secrete IFN-z is related to its developmental

progress and progesterone concentration of the pregnant female (Mann et al., 1999). Low

progesterone concentration in plasma as early as day 6 after insemination has been

implicated as a contributing factor for cows failing to conceive (Bulman and Lamming,

1978; Lukaszewska and Hansel, 1980; Kimura et al., 1987; Lamming and Darwash,

1995; Inskeep, 1995; Mann and Lamming, 1999; Hommeida et al., 2004). Enhanced

luteolytic signals also result from suboptimal progesterone concentrations after

insemination (Mann and Lamming, 1995b). Another approach to increase fertility of

lactating dairy cows has been to directly supplement cows with progesterone. A meta-

analysis of 17 studies revealed that progesterone supplementation after insemination

produced an overall improvement in conception rate of 5% and that the timing of

progesterone supplementation was a critical factor (Mann and Lamming, 1999). One

study revealed depressed conception rates when controlled internal drug releasing

(CIDR) devices containing progesterone were inserted in heifers on day 1 or day 2

following estrus (Van Cleef et al., 1989). In contrast, injection of progesterone (100 mg)

on day 1, 2, 3, and 4 of pregnancy advanced development of conceptuses to 14 days of

gestation in beef cows (Garrett et al., 1988a). These conceptuses had increased length

and secreted a greater array of proteins into medium following a 24 hour culture. When










progesterone supplementation was initiated beginning at day 10 of pregnancy, Macmillan

et al. (1991) found a slight decrease in pregnancy rate (-2.7%), Sreenan and Diskin,

(1983) obtained a small increase (4.3%), and Robinson et al. (1989) obtained a large

increase (29.3%) in pregnancy rate. Villarroel et al. (2004) found that first and second

lactation repeat-breeder Holstein cows were 3.26 times more likely to become pregnant

when cows received progesterone releasing intravaginal device (PRID 1.55g of

progesterone) on day 5 through 19 post-AI.

Inhibition of Luteolysis

The maintenance of a functional CL depends directly upon the intensity of

embryonic signals that attenuates endometrial secretion of PGF2a. Pregnancy fails if an

embryo does not produce sufficient amounts of IFN-z or if production is delayed until

after the critical time-period between days 14 and 17 when the luteolysis would otherwise

occur.

Intrauterine infusions of recombinant bovine IFN-z from days 14 to 24 of the

estrous cycle increased lifespan of the CL and duration of the estrous cycle (Meyer et al.,

1995). Further studies with a large number of cows needs to test whether this treatment

increases pregnancy rates. Co-transfer of embryonic vesicles to increase trophoblastic

signals has been reported to increase pregnancy rates in embryo transfer recipients

(Heyman et al., 1987). Administration of IFN-a by intramuscular injection, which can

also block luteolysis, decreased pregnancy rates in heifers (Barros et al., 1992) because

IFN-a has several adverse actions such as causing hyperthermia (Newton et al., 1990).

Administration of a prostanoid synthesis inhibitor could suppress the luteolytic

stimulus in early pregnancy. Injection of flunixin meglumine (a prostaglandin synthesis

inhibitor) neutralized oxytocin-induced PGF2a TeleaSe, reduced the frequency of short










cycles, and increased pregnancy rate from 33.3% in oxytocin challenged cows to 80% in

oxytocin treated cows that received a flunixin meglumine inj section (Lemaster et al.,

1999). In another study, effects of flunixin meglumine on pregnancy rate were farm or

location dependent (Purcell et al., 2005). Together, these results suggest that certain

conceptuses are unable to inhibit uterine PGF2a Secretion and that reducing prostaglandin

synthesis and stimulating IFN-z secretion could improve pregnancy rates.

Nutritional Strategies

Dairy cows reach peak production on average within the first 4 to 6 weeks after

parturition. Unfortunately, feed and energy intake do not reach maximum levels until

approximately 10 12 weeks postpartum. The end result is a lactating cow with

insufficient nutritional requirements that enters a NEB status.

As mentioned before, energy balance is defined as the difference between energy

gain from feed intake minus the energy expenditure associated with maintenance of

physiological function, growth, and milk production (Staples et al., 1990). Several

studies have reported that negative energy status impaired reproductive performance

(Butler and Smith, 1989; Jorritsma et al., 2000). Different nutritional strategies to

improve energy balance or alter nutrient delivery to improve reproductive function are

described in this section.

Fat feeding to improve energy balance

Fats are glyceride esters of fatty acids that can have a direct effect on the

transcription of genes that encode proteins that are essential to reproductive events

(Mattos et al. 2000). Dietary fats typically increase concentrations of circulating

cholesterol, the precursor of progesterone (Grummer and Carroll, 1991). Ruminants fed









supplemental fat often have a slight increase in blood progesterone concentrations [see

Staples et al. (1998) for review]. Hawkins et al. (1995) suggested that the increase seen

in circulating progesterone when cows are fed supplemental fat was from a reduced rate

of clearance of progesterone rather than an increase in progesterone synthesis. Fat

supplementation has also been shown to stimulate programmed growth of a preovulatory

follicle (Lucy et al., 1993), total number of follicles (Lucy et al., 1991ab; Wehrman et al.,

1991; Thomas and Williams, 1996; Beam and Butler, 1997; Lammoglia, 1997), and size

of preovulatory follicles (Lucy et al., 1990, 1991a, 1993; Beam and Butler, 1997; Oldick

et al., 1997).

Garcia-Boj alil et al. (1998) reported that accumulated plasma progesterone from 0

to 50 days in milk (DIM) was greater, pregnancy rates improved, and energy status did

not change when cows were fed diets of 2.2% calcium salts of fatty acids (CSFA)

compared to non fat-supplemented cows. Similarly, Scott et al. (1995) fed CSFA at 0 or

450 g/d from 1 to 180 or 200 DIM and reported a tendency for CSFA to increase the

proportion of cows exhibiting standing estrus (71.4% vs. 65.6) and a reduction in the

proportion of cows with inactive ovaries.

Other studies have also found a beneficial effect of feeding supplemental fats on

fertility of lactating cows (Erickson et al., 1992; Sklan et al., 1994) while some studies

have found no beneficial effect. Although fertility results are inconsistent when cows

were evaluated after being fed supplemental fat, Staples et al. (1998) suggested that

positive effects (17 percentage unit improvement) are more often reported. When first AI

service and conception or pregnancy rate data was examined, ten studies (Schneider et

al., 1988; Bruckental et al., 1989; Sklan et al., 1989; Armstrong et al., 1990; Ferguson et









al., 1990; Sklan et al., 1991; Garcia-Bojalil, 1993; Scott et al., 1995; Burke et al., 1996;

Son et al., 1996) report an improvement (P < 0. 10) while two studies (Erickson et al.,

1992; Sklan et al., 1994) revealed a strong negative influence accompanied by a large

increase in milk production. Among studies that reported an improvement (Armstrong et

al., 1990; Ferguson et al., 1990; Sklan et al., 1991), a reduced number of services per

conception by feeding a fat supplemented diet occurred as well.

Dietary fats could favor reproductive processes through actions related to energy

balance or through specific actions of individual fatty acids on tissue function. Mattos et

al (2000) has suggested that altered uterine and ovarian function can be mediated through

specific fatty acid precursors in the diet to allow increased steroid and/or eicosanoid

secretion. There are many examples of effects of feeding diets high in specific fatty

acids. Linoleic acid supplemented in the diet prepartum can stimulate arachidonic acid

synthesis and lead to higher concentrations of the series 2 prostaglandins (Thatcher et al.,

1994). It is speculated that linolenic acid may compete with arachidonic acid for binding

sites of a key enzyme, cyclooxygenase 2 (PGHS-2), which is necessary for the synthesis

of PGF2a (Mattos et al., 2000; 2004).

Supplementation of the diet with Hish meal has been reported to reduce uterine

PGF2a Secretion of lactating dairy cows (Thatcher et al., 1997). Fish meal contains

relatively high concentrations of two polyunsaturated fatty acids of the n-3 family, EPA

(eicosapentaenoic acid) and DHA (docosahexaenoic acid). Concentrations of EPA and

DHA in Hish oil have been reported to be 10.8 and 1 1.1% of total fatty acids (Donovan et

al., 2000). EPA and DHA can inhibit secretion of PGFza in different cell culture systems

(Levine and Worth, 1984; Achard et al., 1997) including bovine endometrial cells










(Mattos et al., 2001). Using fish meal to replace soybean meal as a source of protein,

Bruckental et al. (1989) and Armstrong et al. (1990) reported higher pregnancy and

conception rates. These results suggest that high concentrations of EPA and DHA in the

diet can reduce PGF2a endometrial secretion and aid in establishment of pregnancy rates.

Administration of antioxidants

Reactive oxygen species are a possible source of infertility because ovarian

steroidogenic tissue (Carlson et al., 1993; Margolin et al., 1992), spermatozoa (Rivlin et

al., 2004), and preimplantation embryos (Fujitani et al., 1997) become compromised as a

consequence of free radical damage. Vitamin E (i.e., a-tocopherol) and p-carotene are

maj or antioxidants present in plasma membranes of cells (Wang and Quinn, 1999; 2000).

Treatment of cows with vitamin E and selenium can increase the rate of uterine

involution in cows with metritis (Harrison et al., 1986) and improve fertilization rates in

ewes (Segerson and Ganapathy, 1980) and cows (Segerson et al., 1977). In general,

however, treatment of lactating cows with vitamin E alone, through feeding or inj section,

had little or no benefits on postpartum cows (Kappel et al., 1984; Stowe et al., 1988;

Arechiga et al., 1998a; Paula-Lopes et al., 2003).

p-carotene is another cellular antioxidant and is thought to be present at the interior

of membranes or lipoproteins (Niki et al., 1995). Cows fed diets deficient in p-carotene

had lower amounts of progesterone in the CL (Ahlswede and Lotthammer, 1978). In

spite of this, its effect on fertility is controversial. Some authors report benefits of

feeding supplemental p-carotene (Ahlswede and Lotthammer, 1978; Rakes et al., 1985;

Arechiga et al., 1998b) whereas others do not (Wang et al., 1982; Akordor et al., 1986).

There was no strong relationship between serum concentrations of p-carotene and fertility









in dairy cattle (Gossen et al., 2004; Gossen and Hoedemaker, 2005). Injection of vitamin

A, a metabolite of p-carotene, resulted in an increase in the number of recovered

blastocysts from superovulated cows (Shaw et al., 1995).

Crossbreeding

Two bulls (Chief and Elevation) make up about 30% of the gene pool of U. S.

Holsteins (Hansen et al., 2005). As mentioned previously, inbreeding coefficients are

rising in American dairy cattle (Short et al., 1992; Wiggans et al., 1995; Young et al.,

1996; Hansen, 2000; Wall et al., 2005) and there is some evidence that this has

contributed to the decline in fertility seen in dairy cattle (Thompson et al., 2000ab;

Alvarez et al., 2005; Wall et al., 2005). Crossbreeding represents a strategy for

preventing effects of inbreeding especially if the milk yield of crossbreds can approach

that of Holstein cattle.

A study in Canada revealed that some groups of crossbred cattle were equivalent to

Holstein controls in lifetime net profit (McAllister et al., 1994). Hansen et al. (2005)

conducted a study using seven large dairies in California to compare characteristics of

several crossbred animals (Normande-Holstein, Montebeliarde-Hol stein, and

Scandinavian Red-Holstein) versus Holsteins. Milk production as well as fat and protein

production during the first 150 DIM among first lactation cows was not significantly

different among breed types. Holsteins produced an average of 29.9 kg, followed by

Scandinavian Red-Holstein with 29.7 kg, Montebeliarde-Hol stein with 28.8 kg, and

Normande-Holstein with 26.5 kg. Calving difficulty and stillbirths were reduced in

crossbred animals. Survival rates indicate that purebred animals left these dairies sooner.

The first service conception rate was 22% for Holsteins compared to 30 35% for

crossbreds. There were also significantly fewer days open for crossbred cows. Thus,










crossbreeding offers some promise for enhancing fertility. One unanswered question is

the optimal type of mating scheme for the crossbred animals themselves and whether the

resultant loss of heterosis in the F2 animals will reduce any advantage over purebred

cows .

Embryo Transfer

The concept of using embryo transfer (ET) as a tool to increase pregnancy rates is

based on the observation that disruptive events such as anovulation, ovulation of oocytes

with low developmental competence, compromised oviductal transport or uterine

environment, and insemination errors or damaged spermatozoa all occur before the time

when embryos are ordinarily transferred (day 6 8 after estrus) (Hansen and Block,

2004). Selection of morula and blastocyst stage embryos for transfer offers the chance to

avoid pregnancy failure associated with the early stages of embryonic development (day

0 8 after estrus).

It has been proposed that during absence of heat stress, pregnancy rates following

embryo transfer as compared to AI in lactating cows are not optimal (Putney et al.,

1989b; Drost et al., 1994; Ambrose et al., 1997). However, ET may become a more

effective strategy to increase pregnancy rates as compared to AI in lactating cows during

periods of heat stress, and the magnitude of the increased temperature does not seem to

influence overall success following transfer (Hansen and Arechiga, 1999). As embryos

advance in their development, the effects of elevated temperatures become less

significant because embryos become more resistant to the deleterious effects of elevated

temperatures (Ealy et al., 1992; Ealy and Hansen, 1994; Ealy et al., 1995; Edwards and

Hansen, 1997; Rivera and Hansen, 2001). As a result, pregnancy rates following ET









during heat stress are higher than pregnancy rates to AI (Putney et al., 1989b; Ambrose et

al., 1999; Al-Katanani et al., 2002a) although not in the absence of heat stress.

One potential constraint for embryo transfer in lactating cows is the short duration

of estrus and lack of intense mounting activity seen in dairy cows (Dransfield et al.,

1998). This phenomenon is exacerbated by heat stress (Nebel et al., 1997) and will limit

the number of embryos transferred in lactating cows in a program that is dependent upon

estrus detection. The first report of a timed embryo transfer (TET) protocol, where

ovulation was synchronized using an Ovsynch protocol, was by Ambrose et al. (1999)

who evaluated the efficiency of TET using either fresh or frozen-thawed in vitro

produced (IVP) embryos and TAI under heat stress conditions. Pregnancy rates in cows

that received a fresh IVP embryo were higher compared to cows in the TAI group.

Limitations to Optimal Pregnancy Rates Using IVP TET

For ET to replace AI on a wide scale in commercial herds ET must become an

economical breeding alternative and embryos must be inexpensive to produce (Hansen

and Block et al., 2004). Superovulation provides the best source of embryos while the

most likely inexpensive source of embryos will be produced from slaughterhouse oocytes

by IVP since superovulation is costly and requires intensive management and careful

synchronization of the donor cows.

Although embryos produced using IVP systems are relatively inexpensive as

compared to embryos produced by superovulation, pregnancy rates achieved following

transfer of an IVP embryo are often less than what is obtained following transfer of an

embryo produced by superovulation. For example, Hasler (2003) reported a 36.7%

pregnancy rate for in vitro derived embryos vs. 54.8% for in vivo embryos. The reason

for the poor survival of IVP embryos is not known. However, IVP embryos are different









from in vivo embryo in terms of morphology (Massip et al., 1995; Crosier et al., 2001;

Rizos et al., 2002), gene expression (Bertolini et al., 2002a; Lazzari et al., 2002;

Lonergan et al., 2003), metabolism (Krisher et al., 1999; Khurana and Niemann, 2000b)

and chromosomal abnormalities (Iwasaki et al., 1992; Viuff et al., 2000). One or more of

these alterations likely contributes to the poor embryo survival after transfer. Calves born

as the result of in vitro production are also more likely to experience developmental

defects (Hasler et al., 2003; Farin et al., 2006).

One possible strategy for increasing pregnancy rates is to transfer two embryos into

the uterine horn ipsilateral to the CL. This approach is based on the idea that the

likelihood is increased that the cow receives at least one embryo competent for sustained

development. In addition, the transfer of two embryos into the ipsilateral uterine horn to

the CL is likely to increase the amounts of IFN-z and other embryo-derived signaling

molecules in the uterus needed to maintain pregnancy and prevent luteolysis. Co-transfer

of embryonic vesicles to increase trophoblastic signals has been reported to increase

pregnancy rates in ET recipients (Heyman et al., 1987).

In a recent study, there was a tendency for higher calving rates for recipients that

received two embryos in the uterine horn ipsilateral to the CL as compared to recipients

that received one embryo (Bertolini et al., 2002a). The requirement for the antiluteolytic

signal in cattle to be locally administered (del Campo et al., 1977, 1983) means that one

should expect pregnancy rates to be higher in cows that received two embryos in the

same uterine horn (unilateral transfer) than for cows that received two embryos

distributed in both uterine horns (bilateral transfer). The opposite was true for heifers

(Anderson et al., 1979). In other studies, transfer of embryos to create two pregnancies in









the uterine horn ipsilateral to the CL has produced a similar pregnancy rate as bilateral

twins and single pregnancies (Sreenan and Diskin, 1989; Reichenbach et al., 1992) or

reduced pregnancy rate as compared to bilateral transfer (Rowson et al., 1971).

Cryopreservation of IVP Embryos

An additional limitation to the widespread use of IVP embryos in cattle is their

poor survival following cryopreservation. Hasler et al. (1995), Ambrose et al., (1999) and

Al-Katanani et al. (2002a) indicated that IVP embryos do not survive freezing as well as

embryos produced in vivo based on pregnancy rates following transfer as compared to

non-frozen embryos. In vitro survival rates following thawing (Pollard and Leibo, 1993;

Enright et al., 2000; Khurana and Niemann, 2000a; Diez et al., 2001; Guyader-Joly et al.,

1999) and pregnancy rates following thawing and transfer (Hasler et al., 1995; Agca et

al., 1998; Ambrose et al., 1999; Al-Katanani et al., 2002a) are consistently lower for IVP

embryos as compared to embryos produced in vivo by superovulation.

Among the metabolic changes associated with IVP embryos linked to poor

freezability is an increase in lipid content (Abe et al., 1999; Rizos et al., 2002).

Mechanical delipidation (Tominaga et al., 2000; Diez et al., 2001) and addition of

inhibitors of fatty acid synthesis (De la Torre-Sanchez et al., 2005) can improve embryo

survival following cryopreservation. Hatching rates were higher for delipidated embryos

compared to controls when day 7 blastocysts were frozen (Murakami et al., 1998), but

pregnancy rates after the transfer of delipidated embryos was 10.5% compared to 22% for

control embryos (Diez et al., 2001). Although delipidated embryos can survive freezing

conditions when tested in vitro, special consideration must be taken since these embryos

do not reflect higher pregnancies and remain less viable than control embryos.









Manipulating the cryopreservation process to minimize damage to the embryo has

also been considered. Of most promise are procedures based on vitrification, which is

defined as "the solidification of a solution (glass formation) brought about not by

crystallization but by extreme elevation in viscosity during cooling" (Fahy et al., 1984).

Vitrification depends on rapid cooling and thawing of embryos while using high

concentrations of cryoprotectants associated with elevated cooling rates (~2500oC/min,

Palasz and Mapletoft, 1996). Although vitrification does not eliminate toxic effects of

cryoprotectants and osmotic damage, the rapid cooling has been reported to decrease

chilling injury and prevent damage associated with high lipid content (Dobrinsky, 1996;

Martino et al., 1996ab). In vitro survival rates following the thawing of vitrified IVP

embryos was either equal (Van-Wagtendonk et al., 1995) or superior to embryos frozen

conventionally (Dinnyes et al., 1995; Agca et al., 1998; O'Kearney-Flynn et al., 1998).

Sensitivity of in vivo derived embryos to cryopreservation is much less and the

complex environment where the embryo develops is key. It has been reported that

embryos cultured in the sheep oviduct (26%) compared to synthetic oviductal fluid in

culture systems (7%) were better able to tolerate freezing conditions. Embryos cultured

in Buffalo rat liver cells or oviductal cells were more resistant to freezing as well as

compared to embryos not subj ected to co-culture (Massip et al., 1993; Leibo and

Loskutoff, 1993; Tervit et al., 1994).

Summary and Objectives of the Thesis

There has been a precipitous decline in fertility of dairy cows over the last 10-40

years and heat stress is associated with infertility in lactating dairy cows. To characterize

events associated with infertility is important and the purpose of the present series of

experiments described in this thesis was to evaluate strategies that help overcome








reproductive failure. Improving reproductive function in dairy cattle is of maj or interest

and experiments were designed to 1) evaluate strategies for enhancing fertility after AI

using GnRH treatment and 2) further develop ET using IVP embryos as a tool for

increasing fertility by testing whether pregnancy rate could be improved by transfer of

twin embryos and whether the developmental competence of embryos after

cryopreservation could be improved.


4.0



o
3.0 -,



2.0 O


1.5 u


-15


-14.5 f


14 m
I




1.5 ~



2000


1980


1990


Year

Figure 1-1. Rolling herd average (RHA, kg milk per lactation), calving interval (CI), and
services per conception (SPC) for 143 dairy herds continuously enrolled in the
Raleigh DHIA record system from 1970 to 1999 (Lucy, 2001).


9000



8000 .



7000 -



6000



5000
1970













50 s 10500
Cool season
S451 10000

S40-
-9500
co Warm Season Milk Yield
c 35-T
"~ 9000 a,
S30 -1
e 8500 =
o 25- >

a, 20 -1 -8000

.5~~~ 15. 7500


Figure 1-2. Temporal changes in first service pregnancy rate and annual average milk
production from high-producing Holstein-Friesian dairy herds in north-eastern
Spain. Data for pregnancy rate were recorded in the cool (October April
months) and warm season (May-September months). Data were drawn by P.J.
Hansen (unpublished) based on data of Lopez Gatius (2003).















CHAPTER 2
EFFECTIVENESS OF ADMINISTRATION OF GONADOTROPIN RELEASING
HORMONE AT DAY 11, 14 OR 15 AFTER ANTICIPATED OVTULATION FOR
INCREASING FERTILITY OF LACTATINTG DAIRY COWS AND NON-
LACTATINTG HEIFERS

Introduction

One of the approaches proposed to improve fertility in cattle is administration of

GnRH or GnRH analogues at day 1 1-15 after estrus. Inj section of GnRH at this time can

lead to decreased estrogen secretion (Rettmer et al., 1992a; Mann and Lamming, 1995a)

in an action that likely involves luteinization of the dominant follicle (Thatcher et al.,

1989; Rettmer et al., 1992a; Ryan et al., 1994). In some cases, extended estrous cycle

length (Lynch et al., 1999) and increased progesterone secretion also results (Rettmer et

al., 1992a; Stevenson et al., 1993; Ryan et al., 1994; Willard et al., 2003). Improvement

of fertility has been seen by administration of GnRH or its analogues at day 1 1-14 in

nulliparous beef heifers (Rettmer et al., 1992b) and lactating dairy cows (Macmillan et

al., 1986; Lajili et al., 1991; Sheldon et al., 1993; Drew and Peters, 1994; Willard et al.,

2003; L6pez-Gatius et al., 2005a). In contrast to these positive results, there was no

favorable effect of similar treatments of GnRH or GnRH analogues on pregnancy rates in

other studies (Jubb et al., 1990; Stevenson et al., 1993; Ryan et al., 1994; Bartolome et

al., 2005). In a meta-analysis of published results, Peters et al. (2000) concluded that the

overall effect of GnRH administration betweendDay 11 and 14 after anticipated ovulation

was positive, but that results were not consistent between studies.









It is possible that GnRH treatment is more effective at increasing pregnancy rate

per insemination during periods of heat stress than in cool weather because circulating

concentrations of progesterone can be reduced in cows subj ected to heat stress

(Wolfenson et al., 2000). In addition, the anti-luteolytic process may be compromised

because heat stress can decrease growth of the filamentous stage concepts (Biggers et

al., 1987) and increase uterine prostaglandin-Fza Secretion from the uterus (Wolfenson et

al., 1993). Beneficial effects of GnRH treatment at day 1 1-12 after insemination on

fertility have been observed in lactating dairy cows during heat stress (Willard et al.,

2003; L6pez-Gatius et al., 2005a). The purpose of the present series of experiments was

to evaluate the effectiveness of GnRH treatment at either day 11, 14 or 15 after

anticipated ovulation for improving fertility of lactating cows and heifers and determine

whether the beneficial effect of GnRH was greater during summer than winter.

Materials and Methods

Experiment 1 GnRH Administration at Day 11 after Anticipated Ovulation in
Heifers Subjected to Timed Artificial Insemination during Heat Stress

The experiment was conducted at a commercial dairy located in Trenton, Florida

(29037' N 82o49' W) from July to September, 2003 using 149 Holstein heifers. The

heifers ranged in age from 13-23 mo (mean=539 d, SD=76) and ranged in weight from

316 to 448 kg (mean=360 kg, SD=32). Heifers were maintained on grass pasture with

supplemental grass hay. Heifers were randomly allocated to one of four treatments in a 2

x 2 factorial design with main effects of timing of insemination (protocol A vs B) and

treatment (vehicle vs GnRH). The experiment was replicated twice with between 70 and

79 heifers per replicate. Heifers were subj ected to timed artificial insemination (TAI)

based on a protocol published previously (Martinez et al., 2002ab). On Day -10 of the









protocol (Day 0 equals the day of anticipated ovulation), heifers received 100 Cpg (i.m.) of

GnRH (Fertagyl, equivalent to 50 pug /ml gonadorelin diaecetate tetrahydrate; (Intervet

Inc. Millsboro, DE) and an unused intravaginal progesterone-releasing device insert

(EAZI-BREED CIDR" insert, 1.38 g of progesterone, Pfizer Animal Health, New York,

NY, USA). At Day -3, CIDR devices were removed and 25 mg (i.m.) of prostaglandin

Fz, (PGF2,; 5 ml Lutalyse", Pfizer Animal Health, New York, NY, USA) was

administered. A second 100 Cpg GnRH injection was given 48 h after CIDR withdrawal

(Day -1). Regardless of estrus behavior, heifers in protocol A were inseminated 24 h

after the second GnRH injection (d 0) and heifers in protocol B were inseminated at the

same time as the second GnRH injection (d -1). Two individuals conducted all

inseminations and semen from one sire was used for all heifers. Heifers from each

synchronization treatment protocol were randomly allocated to receive either 100 Cpg of

GnRH, (i.m.) or an equivalent volume (2 ml) of vehicle (9 mg/ml of benzyl alcohol and

7.47 mg/ml of sodium chloride in water) at Day 11 after anticipated ovulation.

On the day of insemination and on Day 11 after anticipated ovulation, a 10-ml

blood sample was collected via coccygeal or jugular venipuncture into heparinized tubes

(Becton Dickinson, Franklin Lakes, NJ) to measure the proportion of heifers successfully

synchronized. An animal was considered synchronized if progesterone concentrations

were lower than 1 ng/ml on the day of insemination and greater than 1 ng/ml on Day 11

after anticipated ovulation. A third blood sample was collected in a subset of 76 heifers

at Day 15 after anticipated ovulation (i.e., 4 d after the inj section of GnRH or vehicle) to

determine the effect of GnRH treatment on serum concentrations of progesterone.

Pregnancy was diagnosed by palpation per rectum at Day 44-51 after insemination.









Blood samples were stored on ice (~2-4 h) until centrifugation at 2,000 x g for 20

min at 4 oC to obtain plasma. Plasma was stored at -20 oC until assayed for progesterone

concentrations using a progesterone radioimmunoassay kit (Coat-a-Count@; Diagnostic

Products Corp., Los Angeles, CA). The sensitivity of the assay was 0.1 ng/ml and the

intrassay and interassay CV were each 6%.

Experiment 2 GnRH Administration at Day 11 after Anticipated Ovulation in
Lactating Cows Subjected to Timed Artificial Insemination

This study took place at the University of Florida Dairy Research Unit (Hague,

Florida; 29046' N 82o25' W). A total of 244 primiparous and multiparous lactating

Holstein cows housed in freestall barns equipped with a fan-and-sprinkler system were

used. Cows were fed a total mixed ration (TMR) to meet or exceed requirements

recommended for lactating dairy cows, were milked three times a day, and received

bovine somatotropin (Posilac, Monsanto Corp., St. Louis, MO) according to

manufacturer' s recommendation. Cows were subj ected to the OvSynch TAI program

(Schmitt et al., 1996a; Pursley et al., 1998); 100 Cpg (i.m.) GnRH (Fertagyl equivalent to

50 Cpg /ml gonadorelin diaecetate tetrahydrate, Intervet, Millsboro, DE) was inj ected at

Day 0 of the protocol, 25 mg (i.m.) PGF2, (5 ml of Lutalyse", Pfizer Animal Health, New

York, NY, USA) was given at Day 7, 100 Cpg (i.m.), GnRH was again injected, i.m., at

Day 9, and cows were inseminated 16 h later (the day of anticipated ovulation). At the

time of insemination (from January September, 2004), 244 cows were between 76 and

594 days in milk (DIM; mean= 176, SD= 114). Multiple individuals conducted

inseminations (n=7) and multiple AI sires were used (n=45).

Cows were randomly assigned within pair to receive 100 Cpg (i.m.) GnRH or an

equivalent volume (2 ml) of vehicle (9 mg/ml benzyl alcohol and 7.47 mg/ml sodium









chloride in water) at Day 11 after anticipated ovulation (i.e., 11 d after insemination).

Rectal temperature was recorded in a subset of cows (n=134) on the afternoon of Day 1 1

after TAI at 1500 1600 h. Pregnancy was diagnosed by rectal palpation at ~Day 46

after insemination.

Experiment 3 GnRH Administration at Day 14 after Anticipated Ovulation in
Lactating Cows Subjected to Timed Artificial Insemination

This study was conducted at two different locations using lactating Holsteins.

Farm 1 was the University of Florida Dairy Research Unit at Hague, Florida while farm 2

was a commercial dairy in Chiefland, Florida (29030' N 82o52' W). Cows from farm 1

(n=307) were inseminated from February November 2004 and cows in farm 2 (n=170)

were inseminated from June October 2004. At both farms, primiparous and multiparous

cows were used. At farm 1, 307 cows were TAI between 76 590 DIM (mean= 187,

SD= 102). Multiple individuals conducted inseminations (n=7) and multiple AI sires

were used (n=42). At farm 2, 170 cows were used for first service after calving using

seven different sires and one inseminator. The TAI protocol was designed to achieve

insemination at 60 + 3 d in milk. Cows in both farms were housed in freestall barns

equipped with fans and sprinklers, were fed a TMR, were milked three times a day, and

received Posilac@ (Monstanto, St. Louis, MO) according to manufacturer' s directions.

Cows in farm 1 were subjected to an OvSynch protocol as described for

Experiment 2. Cows for farm 2 were subj ected to a TAI protocol that incorporated a pre-

synchronization with PGF2, (Moreira et al., 2001) and the CIDR-Synch ovulation

synchronization protocol (Portaluppi and Stevenson, 2005). Cows received two

inj sections of 25 mg PGF2, (i.m.) (Lutalyse) 14 d apart starting on Day 21-27 DIM.

Twelve days after the second PGF2, inj section, a timed ovulation synchronization protocol









was initiated. Cows received 100 Cpg (i.m.) GnRH (2 ml of Cystorelin ; Merial Limited,

Iselin, NJ, USA) and an unused EAZI-BREED CIDR" intravaginal progesterone-

releasing device insert. Seven days later, CIDR devices were removed and 25 mg (i.m.)

PGF2, waS given. Cows received a second 100 Cpg (i.m.) injection of GnRH at 72 h after

CIDR withdrawal. Estrus was detected using tail chalk or KaMar estrus detection

patches (KAMAR Inc., Steamboat Springs, CO, USA). Cows observed in estrus at 24 or

48 h after CIDR removal were inseminated at estrus. Cows not observed in estrus were

inseminated at 72 h after CIDR withdrawal. Ovulation was anticipated to occur 72 h

after CIDR withdrawal. All animals received the GnRH inj section at 72 h regardless of

estrus behavior. Cows were also randomly assigned within pair to receive either 100 Cpg

(i.m.) GnRH (2 ml of Cystorelin ; Merial Limited, Iselin, NJ, USA), or vehicle (as for

experiment 2) at 14 d after anticipated ovulation. Pregnancy was diagnosed by rectal

palpation at ~Day 45 after insemination.

Rectal temperature was recorded in a subset of 100 cows in Farm 1 and 39 cows in

Farm 2 at 1500 h of Day 14 after anticipated ovulation.

Experiment 4 GnRH Administration at Day 14 after Anticipated Ovulation in
Lactating Cows Subjected to Timed Artificial Insemination During Heat Stress

This study took place at the University of Florida Dairy Research Unit with

inseminations in April to June, 2005. A total of 137 primiparous and multiparous

lactating Holstein cows ranging in DIM from 78 to 566 d (mean= 185, SD= 110) were

subj ected to an OvSynch protocol as described for Experiment 2. Multiple individuals

conducted inseminations (n=4) and multiple AI sires were used (n=22).

Cows were randomly assigned within pair to receive 100 Cpg (i.m.) GnRH or an

equivalent volume (2 ml) of vehicle (9 mg/ml benzyl alcohol and 7.47 mg/ml sodium









chloride in water) at Day 14 after anticipated ovulation (i.e., 14 d after insemination).

Pregnancy was diagnosed by rectal palpation at ~Day 46 after insemination.

Experiment 5 GnRH Administration at Day 14 or Day 15 after Detected Estrus

This study took place at a commercial dairy in Chiefland, Florida. A total of 296

primiparous and multiparous lactating Holstein cows inseminated at detected estrus were

used. Cows were inseminated from April August, 2005. At the time of insemination,

cows were between 51 and 235 DIM (mean= 122, SD= 40).

Estrus was detected using tail chalk or KaMar estrus detection patches (KAMAR

Inc., Steamboat Springs, CO, USA). Estrus detection patches were visually monitored

twice (morning and afternoon) daily by the inseminator. When cows were first diagnosed

in estrus in the afternoon, insemination was performed the next morning. When estrus

was first detected in the morning, cows were inseminated at that time. Cows were bred

by one inseminator and 3 1 different sires used. Every other day of the experiment, cows

were selected to receive injections at Day 14 or 15 after insemination. Within each day,

cows were randomly assigned within a pair to receive 100 Cpg (i.m.) GnRH or an

equivalent volume (2 ml) of vehicle (9 mg/ml benzyl alcohol and 7.47 mg/ml sodium

chloride in water). Pregnancy was diagnosed by rectal palpation at ~Day 45 after

insemination.

Statistical Analysis

Data on pregnancy rate were analyzed by logistic regression with the LOGISTIC

and GENMOD procedures of SAS (SAS for Windows, Release 8.02; SAS Inst., Inc.,

Cary, NC). For the LOGISTIC procedure, a backward stepwise logistic model was used.

Variables were continuously removed from the model by the Wald statistic criterion if

the significance was greater than 0.20. The Wald X2 statistic was used to determine the










significance of each main effect that remained in the reduced model. The adjusted odds

ratio (AOR) estimates and the 95% Wald confidence intervals from logistic regression

were obtained for each variable that remained in the final statistical model following the

backward elimination. Data were also analyzed by PROC GENMOD and P values for

significant treatment effects are reported from this analysis. The full mathematical model

for experiment 1 included main effects of inseminator, treatment, protocol, replicate,

replicate x protocol, replicate x treatment, replicate x inseminator, protocol x treatment,

protocol x inseminator, treatment x inseminator. The full mathematical model for

experiment 2 included the effects of season of insemination (January to March vs April to

September), treatment, and season x treatment. For experiment 3, the full mathematical

model included the effects of farm, treatment, season of insemination (warm vs cool

season; farm 1 = October to March vs April to September; farm 2 = June to September vs

October to November), and season x treatment, season x farm, and treatment x farm. In

addition, a subset of data composed of cows from farm 2 only was analyzed where the

additional factor of estrus detection (yes or no) was included in the model. For

experiment 4, the full mathematical model included the effects of treatment, month of

insemination, parity (1 vs others), sire, DIM at insemination class (<150 d vs > 150 d),

parity x treatment, DIM class x treatment and month x treatment. For experiment 5, the

full mathematical model included the effects of treatment, season of insemination (April

and May vs June to August), parity (1 vs > 1), number of services (1, 2 and >2), DIM at

insemination class (<150 d vs > 150 d) and interactions of main effects with treatment.

Since interactions were not significant, data were reanalyzed with main effects only.









Data on rectal temperatures were analyzed by least-squares analysis of variance

using the GLM procedure of SAS. The model included effects of season (Exp.2) or

season, farm and farm x season (Exp. 3).

A meta-analysis was performed using Mantel-Haenszel procedures available using

software downloaded from http://www.pitt.edu/~superl/lecture/lecl 171/index.htm.

Three analyses were performed using all experiments, experiments with GnRH

treatment at Day 11, and experiments with GnRH treatment at Day 14 or 15.

Results

Experiment 1 GnRH Administration at Day 11 after Anticipated Ovulation in
Heifers Subjected to Timed Artificial Insemination During Heat Stress

Based on progesterone concentrations measured at insemination and at Day 11 after

anticipated ovulation, estrous cycles of 137/149 (92%) of the heifers were successfully

synchronized. Pregnancy rate was not significantly affected by GnRH treatment or

insemination protocol. This is true whether all heifers were considered (Table 1) or only

those successfully synchronized (results not shown). There was also no effect (P > 0.10)

of GnRH treatment at Day 11 on concentrations of plasma progesterone on Day 15.

Values were 3.5 & 0.19 ng/ml for heifers receiving vehicle and 3.6 & 0.19 ng/ml for

heifers receiving GnRH.

Experiment 2 GnRH Administration at Day 11 after Anticipated Ovulation in
Lactating Cows Subjected to Timed Artificial Insemination

Treatment with GnRH did not significantly (P > 0.10) affect pregnancy rate per

insemination (Table 2). This was true for inseminations in both cool seasons (January to

March) and warm season (April to September) (results not shown). There was also no

significant difference in pregnancy rate between seasons.









Rectal temperatures were higher (P < 0.001) for cows in the warm season (least-

squares means + SEM; 39.3 + 0.07 oC) than for cows in the cool season (38.9 + 0.07 oC).

Experiment 3 GnRH Administration at Day 14 after Anticipated Ovulation in
Lactating Cows Subjected to Timed Artificial Insemination

Inj section of GnRH increased pregnancy rates at both farms (treatment, P < 0.02;

treatment x farm, non-significant) (Table 3). While pregnancy rates were lower in

summer than winter (P < 0.05), the effect of GnRH was apparent in both seasons and the

season x treatment interaction was not significant.

Cows in farm 2 were monitored for estrus. No cows were seen in estrus at 24 h

after PGF2a, 4.7% (8/171) were detected in estrus at 48 h, 32.2% (55/171) at 72 h, and

63.1% (108/171) were not detected in estrus. Cows in estrus at 48 h were inseminated at

that time while other cows (those seen in estrus at 72 h and those not seen in estrus) were

inseminated at 72 h. There was an estrus detection class (detected in estrus vs not

detected) x treatment interaction (P < 0.03) on pregnancy rate per insemination that

reflected the fact that GnRH was effective at increasing pregnancy rate for those cows

displaying estrus [3/29 (10%) for control and 12/34 (3 5%) for GnRH[ but had no effect

for those cows not displaying estrus [7/54 (13%) for control and 4/54 (8%) for GnRH].

Rectal temperatures were higher (P < 0.01) for cows in the warm season (least-

s uares means + SEM: 39.4 + 0.06 oC) than for cows in the cool season (39. 1 + 0. 11 oC)

and hi her (P < 0.001) for farm 2 (39.5 + 0.10 oC) than for farm 1 (39.1 + 0.07 oC but

there was no farm x season interaction.

Experiment 4 GnRH Administration at Day 14 after Anticipated Ovulation in
Lactating Cows Subjected to Timed Artificial Insemination During Heat Stress

Treatment with GnRH did not significantly affect pregnancy rate (Table 4).

Pregnancy rate was higher (P<0.02) for cows inseminated at or before 150 DIM (30.3%,









20/66) than for cows inseminated after 150 DIM (12.7%, 9/71). There were no other

significant main effects or interactions of GnRH treatment with other effects.

Experiment 5 GnRH Administration at Day 14 or Day 15 after Detected Estrus

Overall, pregnancy rate was higher (P<0.0001) for cows inseminated in April and

May (55/171, 32.2%) than for animals inseminated in June, July or August (12/125,

9.6%). There were, however, no other significant main effects or interactions of GnRH

treatment with other effects. Pregnancy rates were 25.6% (32/125) for cows receiving

vehicle at day 14 or 15, 20.7% (19/92) for cows receiving GnRH at Day 14, and 20.3%

(16/79) for cows receiving GnRH at Day 15.

Overall Effectiveness of GnRH Treatment as Determined by Meta-Analysis

When data from multiple experiments were considered together by meta-analysis,

there was no significant effect of GnRH on pregnancy rate. This was the case when all

experiments were considered (odds ratio=0.97; 95% CI=0.63, 1.50), or whether

experiments with GnRH treatment on Day 11 (odds ratio=0.87; 95% CI=0.50, 1.50) or

Day 14 or 15 (odds ratio=1.06; 95% CI=0.68, 1.65) were considered separately.

Discussion

Overall, there was no significant effect of GnRH treatment on pregnancy rate. In

particular, GnRH treatment at Day 11 after anticipated ovulation did not improve

pregnancy rate of heifers or lactating cows in any experiment, whether animals were

exposed to heat stress or not. Moreover, GnRH did not consistently improve fertility

when given at Day 14 after anticipated ovulation or at Days 14 or 15 after insemination.

In one experiment (experiment 3), administration of GnRH at Day 14 after anticipated

ovulation in cows subjected to TAI increased pregnancy rate of lactating cows in









summer and winter at two locations. However, this positive effect could not be replicated

either in lactating cows subj ected to TAI or for cows inseminated at standing estrus.

The variability in response to GnRH is reminiscent of the results of the meta-

analysis of published studies performed by Peters et al. (2000) in which inconsistency

between studies was noted. Variability in results could reflect either error in estimates of

treatment effects because of small numbers of experimental units or variability in

biological responses to GnRH. The number of animals used for the present studies varied

and could have been too small in some studies to detect significant differences or have

lead to sampling errors that obscured the magnitude or direction of the treatment

differences. However, meta-analysis of the entire data set, involving 1303 cows,

indicated that there was no overall effect of GnRH.

It is also possible that herds differ between each other or over time in the

predominant biological response to GnRH treatment. Presumably, beneficial effects of

GnRH post-insemination on fertility are related to its actions to cause LH release.

Treatment with GnRH at Day 1 1-15 of the estrous cycle can decrease function of the

dominant follicle (Thatcher et al., 1989; Rettmer et al., 1992a; Ryan et al., 1994; Mann

and Lamming, 1995a) and increase progesterone secretion (Rettmer et al., 1992a;

Stevenson et al., 1993; Ryan et al., 1994; Willard et al., 2003). The reduction in

estradiol-17P secretion caused by GnRH should delay luteolysis and conceivably allow a

slowly-developing concepts additional time to initiate secretion of interferon-z. Low

progesterone secretion may also compromise fertility in dairy cattle (Mann and

Lamming, 1999; Lucy, 2001) and an increase in progesterone secretion caused by GnRH

may facilitate embryonic development. Whether a herd responds to GnRH by undergoing









follicular changes may depend upon the characteristics of follicular growth because a

follicle must reach 10 mm in diameter to ovulate in response to LH (Sartori et al., 2001).

Perhaps, herds that do not respond to GnRH with an increase in fertility are herds where

many cows have lower follicular growth or follicular wave characteristics that do not

result in sufficient follicular development at the time of inj section.

One example of the potential importance of follicular dynamics in determining

responses to GnRH is the expected response to GnRH treatment at Day 11 after

anticipated ovulation. In the current studies, injection of GnRH at Day 11 after

anticipated ovulation did not increase pregnancy rates in either lactating Holstein cows or

nulliparous heifers. For lactating cows, the absence of an effect of GnRH at Day 11 was

seen in both summer and winter. This result, which agrees with other studies in which

inj section of GnRH at Day 11 does not affect fertility (Stevenson et al., 1993; Jubb et al.,

1990), is in contrast to other studies indicating that GnRH treatment at Day 11 can

increase fertility of heifers (Rettmer et al., 1992b) and lactating cows (Sheldon and

Dobson, 1993; Willard et al., 2003). One factor that could influence the effectiveness of

GnRH treatment at Day 11 is the number of follicular waves that an individual animal

expresses. Animals with estrous cycles characterized by three follicular waves have

larger second-wave dominant follicles at Day 11 of the estrous cycle than animals with

two-wave cycles (Ginther et al., 1989; Savio et al., 1990; Ko et al., 1991) and thus the

preponderance of cycle type (two-wave vs three-wave) within a herd may determine

effectiveness of GnRH treatment at Day 11. There is variation from study to study in the

relative frequency of three-wave vs two-wave cycles, at least among Holstein heifers

(Ginther et al., 1989; Knopf et al., 1989; Rajamahendran et al., 1991; Gong et al., 1993),










and this variation is evidence for herd-to-herd variation in frequency of follicular wave

patterns.

Even in animals with three-wave follicular cycles, Day 11 would appear to not be

an optimal time of the estrous cycle for using GnRH to cause luteinization because the

second-wave dominant follicle is smaller at Day 11 than at 14-15 in heifers (Ginther et

al., 1989; Ko et al., 1991) and lactating cows (Ko et al., 1991). Results from a limited

number of cows in Experiment 3 suggested that the effectiveness of GnRH at Day 14

after anticipated ovulation depends upon whether cows are detected in estrus.

Presumably, ovulation occurred on average sooner for cows in estrus at 48 and 72 h after

prostaglandin than for cows not detected in estrus (which contains cows that had not

initiated estrus by 72 h as well as some cows in which estrus occurred by 72 h but was

not detected). Among those detected in estrus, GnRH injection improved fertility from

10.3% to 35.3%. Among animals not detected in estrus, however, there was no

difference in pregnancy rate between animals treated with vehicle (13.0%) or GnRH

(7.6%). It is likely that GnRH did not affect pregnancy rate in the cows not detected in

estrus because this group included cows that were anovulatory at insemination or that

were not successfully synchronized; GnRH would be unlikely to increase pregnancy rate

in these animals.

It was hypothesized that beneficial effects of GnRH would be greater during heat

stress because this condition can decrease growth of the filamentous stage concepts

(Biggers et al., 1987), increase uterine prostaglandin Fza Secretion from the uterus

(Wolfenson et al., 1993) and reduce circulating concentrations of progesterone

(Wolfenson et al., 2000). Beneficial effects of GnRH treatment at Day 11-12 after









insemination on fertility have been observed in lactating dairy cows during heat stress

(Willard et al., 2003; L6pez-Gatius et al., 2005a). There was no evidence, however, that

GnRH was more effective during the summer. In particular, the increase in pregnancy

rate caused by inj section of GnRH at Day 14 during experiment 3 was similar for cows

inseminated in summer and winter. In other experiments conducted during the summer,

GnRH was without beneficial effect.

In experiment 1, there were no differences in pregnancy rates for Holstein heifers

inseminated either at second GnRH inj section (24.4%) or 24 after GnRH (19.8%). This

result is similar to results of Pursley et al. (1998) who reported little difference in

pregnancy rates and no differences in calving rates between lactating cows inseminated at

0, 8, 16, or 24 h after the second GnRH inj section of the OvSynch regimen. The

pregnancy rates achieved with heifers in experiment 1 were low compared to other

studies in which heifers received a similar ovulation synchronization program (Martinez

et al., 2002ab). The low fertility was not a result of delayed puberty or unresponsiveness

to the synchronization protocol because 92% of the heifers had both low progesterone

concentrations during the expected periovulatory period and high progesterone

concentrations at the predicted luteal phase of the cycle. It is possible that some of these

heifers classified as synchronized experienced short estrous cycles (Schmitt et al., 1996b;

Moreira et al., 2000a). The experiment was conducted during the summer and it is also

possible that heat stress reduced fertility. Although fertility in Holstein heifers does not

always decline during the summer (Ron et al., 1984; Badinga et al., 1985), there is one

report (Donovan et al., 2003) that heifers from a dairy farm in north central Florida

inseminated in summer were more than four times less likely to become pregnant to first





































GnRH Treatment
GnRH 20/78 25.6 1.29 0.59 -2.83 0.41
Vehicle 14/71 19.7


Protocol 4
B 20/79 25.3 1.34 0.61 -2.95 0.41
A 14/70 20.0


insemination than heifers inseminated during the rest of the year. It is also possible that

the one sire used to inseminate all heifers was not a fertile bull.

In conclusion, inj section of GnRH at Day 11-15 after anticipated ovulation or

insemination did not consistently increase pregnancy rates in heifers or lactating cows.

The fact that GnRH administration was effective in one study indicates that such a

treatment may be useful for increasing pregnancy rate in some herds or situations. More

work will be required to describe factors that could identify which groups of cows would

be most likely to benefit from GnRH treatment.

Table 2-1. Descriptive statistics, adjusted odds ratio (AOR) estimates, and 95% Wald
confidence intervals (CI) for effect of GnRH administration at Day 11 after
anticipated ovulation and ovulation synchronization protocol on pregnancy
rates of heifers during heat stress.

Pregnancy rate
Proportion % AOR 95% Wald CI P-value 2


Data represent the number of females pregnant at Day 44-5 1 after insemination / total number
of females inseminated.
2 Derived from PROC GENMOD.
3 Wald chi-square statistic =0.54 (N.S).
4 Wald chi-square statistic = 0.40 (N.S.)










Table 2-2. Descriptive statistics, adjusted odds ratio (AOR) estimates, and 95% Wald
confidence intervals (CI) for effect of GnRH administration at Day 11 after
anticipated ovulation and season of insemination on pregnancy rates of
lactating cows subj ected to timed artificial insemination.

Pregnancy rate
Proportion % AOR 95% Wald CI P-value 2
GnRH Treatment
GnRH 26/121 21.5 0.66 0.37 -1.18 0.16
Vehicle 36/123 29.3

Season 4
January March 30/103 29.1 1.38 0.77 -2.48 0.27
April September 32/141 22.7
'Data represent the number of females pregnant at ~d 45 after insemination / total number of
females inseminated.
SDerived from PROC GENMOD.
2Wald chi-square statistic =1.50 (N.S).
4 Wald chi-square statistic = 1.38 (N.S.)

Table 2-3. Descriptive statistics, adjusted odds ratio (AOR) estimates, and 95% Wald
confidence intervals (CI) for effect of GnRH administration at Day 14 after
anticipated ovulation and season of insemination on pregnancy rates of
lactating cows subj ected to timed artificial insemination.

Pregnancy rate
Proportion % AOR 95% Wald CI P-value 2
GnRH Treatment
GnRH 49/241 20.3 1.76 1.07 -2.89 0.02
Vehicle 3 0/23 6 12.7

Season 4
Oct, Nov, Feb,
40/187 21.4 1.76 1.08 -2.87 0.02
March
May September 39/290 13.5
Data represent the number of females pregnant at ~Day 45 after insemination / total number of
females inseminated.
SDerived from PROC GENMOD.
SWald chi-square statistic =4.94 (P=0.026).
4 Wald chi-square statistic = 5.12 (P=0.024)









Table 2-4. Descriptive statistics, adjusted odds ratio (AOR) estimates, and 95% Wald
confidence intervals (CI) for effect of GnRH administration at Day 14 after
anticipated ovulation and Days in milk (<150 d vs > 150) at insemination on
pregnancy rates of lactating cows subj ected to timed artificial insemination
during heat stress.

Pregnancy rate
Proportion % AOR 95% Wald CI P-value 2
GnRH Treatment
GnRH 11/73 15.1 0.43 0.18 -1.04 0.05
Vehicle 18/64 28.1


Days in milk at
insemination
< 150 d 20/66 30.3 3.11 1.27 7.62 0.02
> 150 d 9/71 12.7


SData represent the number of females pregnant at
number of females inseminated.
2 Derived from PROC GENMOD.
3 Wald chi-square statistic =3.55 (P=0.060).
4 Wald chi-square statistic = 6.12 (P=0.013)


-Day 45 after insemination / total















CHAPTER 3
EFFECT OF TRANSFER OF ONE OR TWO INT VITRO-PRODUCED EMBRYOS
AND POST-TRANSFER ADMINISTRATION OF GONADOTROPIN RELEASING
HORMONE ON PREGNANCY RATES OF HEAT-STRESSED DAIRY CATTLE

Introduction

The in vitro produced (IVP) embryo is different from the embryo produced in vivo

in terms of morphology (Iwasaki et al., 1992; Massip et al., 1995; Crosier et al., 2001),

gene expression (Bertolini et al., 2002a; Lazzari et al., 2002; Lonergan et al., 2003),

metabolism (Khurana et al., 2000b), and incidence of chromosomal abnormalities

(Iwasaki et al., 1992; Viuff et al., 2000). Not surprisingly, pregnancy rates achieved

following transfer of an IVP embryo are often less than what is obtained following

transfer of an embryo produced by superovulation and calves born as the result of in vitro

production are more likely to experience developmental defects (Hasler et al., 2003).

Problems associated with the transfer of IVP embryos have limited the realization of the

potential of these embryos for enhancing genetic improvement and reproductive

performance of lactating dairy cattle (Rutledge, 2001; Hansen and Block et al., 2004).

One method that might be useful for increasing pregnancy rates in dairy cattle

recipients that receive an IVP embryo is to transfer two embryos into the uterine horn

ipsilateral to the CL. Such a treatment might increase pregnancy rate because the

likelihood is increased that the cow receives at least one embryo competent for sustained

development. In addition, the transfer of two embryos into the ipsilateral uterine horn is

likely to increase the amounts of interferon-z and other embryonic signaling molecules in

the uterus needed to maintain pregnancy and prevent luteolysis. Co-transfer of










embryonic vesicles to increase trophoblastic signals has been reported to increase

pregnancy rates in embryo transfer recipients (Heyman et al., 1987). For the current

experiment, both embryos were transferred into the uterine horn ipsilateral to the CL

because of the requirement for the antiluteolytic signal in cattle to be locally administered

(Del Campo et al., 1977; 1983). In a recent study with a small number of transfers (n=10

to 28 recipients), there was a tendency for higher calving rate for recipients that received

two embryos in the uterine horn ipsilateral to the CL as compared to recipients that

received one embryo (Bertolini et al., 2002b). Anderson et al. (1979) found a tendency

for pregnancy rates to be higher in cows that received two embryos in the same uterine

horn (unilateral transfer) than for cows that received two embryos distributed in both

uterine horns (bilateral transfer); the opposite was true for heifers. In other studies,

transfer of embryos to create two pregnancies in the uterine horn ipsilateral to the CL has

produced a similar pregnancy rate as bilateral twins and single pregnancies (Sreenan and

Diskin, 1989; Reichenbach et al., 1992) or reduced pregnancy rate as compared to

bilateral transfer (Rowson et al., 1971).

Another treatment that has potential for increasing pregnancy rates in embryo

transfer recipients is inj section of GnRH at Day 11 after the anticipated day of ovulation.

Such a treatment was shown to increase pregnancy rates in heat-stressed, lactating cows

following insemination (Sheldon and Dobson, 1993; Willard et al., 2003) and embryo

transfer (Block et al., 2003). Treatment with GnRH or its analogues at Day 11 tol2 of

the estrous cycle has been reported to increase progesterone secretion (Ryan et al., 1994;

Willard et al., 2003) and inhibit function of the dominant follicle (Savio et al., 1990;

Ryan et al., 1994) to possibly delay luteolysis.










The purpose of the current pair of experiments was to examine the effectiveness of

unilateral transfer of twin embryos and treatment with GnRH at Day 11 after the

anticipated day of ovulation for increasing pregnancy rates in dairy cattle recipients that

received IVP embryos. Experiments were performed during periods of heat stress

because embryo transfer offers benefits as a method for increasing pregnancy rate as

compared to AI in females subj ected to heat stress (Rutledge, 2001).

Materials and Methods

Experiment 1 Single or Twin Transfer of IVP Embryos into Crossbred Dairy
Recipients

The experiment was conducted at a commercial dairy located in Santa Cruz,

Bolivia (17048' S, 63"10' W) from November December, 2004. Data on minimum and

maximum air temperatures during the experiment collected by Servicio Nacional de

Meteorologia e Hidrologia (http://www.senamhi. gov.bo/meteorologia/) for Santa Cruz

are presented in Figure 1. Females receiving embryos included 32 virgin crossbred

heifers sired by Simmental, Gyr, or Brown Swiss bulls and Holstein or Holstein

crossbred dams and 26 lactating, crossbred cows with the proportion of Holstein varying

from 1/2 to 15/16. The heifers ranged in age from 363 to 2070 d (mean = 850 d and

median = 664 d; SD = 421 d) and ranged in weight from 247 to 430 kg (mean = 310 kg

and median = 288 kg; SD = 52.3 kg). Animals were maintained on grass pasture until

two weeks prior to the start of the synchronization program when they also received a

supplement of 6 kg/head/d of spent brewers' grain. The cows ranged in age from 820 to

4075 d (mean = 2083 d and median = 1670 d; SD = 986 d), were maintained on grass

pasture, and received 11 kg of brewers' grains and 2 kg of a soybean-based concentrate

mixture before each milking. Cows were milked two times per day and ranged from 110









to 417 d in milk (mean =190 d and median = 170 d; SD = 75 d). Milk yield per day

across all days of lactation ranged from 5.9 to 21.1 kg/d (mean = 12.5 kg/d and median =

12.6 kg/d; SD = 3.8 kg/d).

Recipients were synchronized for timed embryo transfer using a modified OvSynch

protocol (Portaluppi and Stevenson, 2005) with the inclusion of a controlled intravaginal

drug releasing device (EAZI-BREED CIDR" insert, 1.38 g of progesterone, Pfizer

Animal Health, New York, NY, USA). On Day -10 (Day 0 equals the day of anticipated

ovulation), females received 100 Cpg (i.m.) of GnRH (1 ml of Profertil@; Tortuga Cia.

Zootecnica Agraria, Sho Paulo, Brazil) and an intravaginal progesterone-releasing device

insert that had been used one time previously. On Day -3, CIDR devices were removed

and females received 150 Cpg (i.m.) of PGFz, (2 ml of Prostaglandina Tortuga, Tortuga

Cia. Zootecnica Agraria). On Day 0, 100 Cpg (i.m.) of GnRH was administered.

Behavioral symptoms of estrus were monitored about 5 times each day for 3 d following

CIDR removal and PGF2, inj section. On Day 6 after anticipated ovulation, all females,

including those not seen in estrus, were examined per rectum for the presence of a CL

using an Aloka 210 ultrasound unit equipped with a 5 MHz linear array probe (Aloka,

Wallingford, CT, USA). A group of females having a CL (n=32 heifers and n=26 cows)

were randomly selected within recipient type (heifers or cows) to receive one (n=31

females) or two (n=27 females) embryos on Day 7 after anticipated ovulation. For

embryo transfer, an epidural block of 5 ml of lidocaine hydrochloride (2% w/v; Sparhawk

Laboratories Inc., Lenexa, KS, USA) was administered to each recipient, and one or two

IVP embryos were deposited into the uterine horn ipsilateral to the ovary containing the

CL. One technician conducted all transfers.









A total of 85 blastocysts (72 at Day 7 after insemination and 13 and Day 8 after

insemination) were transferred in this experiment. Of these, six were produced by

Transova (Sioux City, IA, USA) using Holstein oocytes and a Holstein sire and were

cultured in Synthetic Oviductal Fluid (SOF) medium. Embryos were shipped overnight

in a portable incubator to Gainesville, FL, USA on Day 4 after insemination. Embryos

were transferred to fresh microdrops of a modified SOF (Fischer-Brown et al., 2002)

prepared by Specialty Media (Phillipsburg, NJ, USA) and cultured at 38.5oC in a

humidified atmosphere of 5% 02 and 5% (v/v) CO2 (balance N2). The remainder were

produced using oocytes obtained from ovaries of a variety of breeds collected at a local

abattoir located at a travel distance of approximately 1.5 h from the Gainesville

laboratory. Procedures, reagents, and media formulation for oocyte maturation,

fertilization, and embryo culture were as previously described (Roth and Hansen, 2005)

with some modifications. Cumulus-oocyte complexes were matured for approximately

22 h at 38.50C in an atmosphere of 5% (v/v) CO2 in humidified air and then inseminated

with a cocktail of Percoll-purified spermatozoa from three different bulls of various

breeds. At 8 12 h post-insemination (hpi), putative zygotes were denuded of cumulus

cells by suspension in Hepes-TALP medium (Caisson, Rexburg, ID, USA) containing

1000 units/ml hyaluronidase type IV (Sigma, St Louis, MO, USA) and vortexed in a

microcentrifuge tube for 5 min. Presumptive zygotes were then placed in groups of ~30 in

50 Cll microdrops of KSOM-BE2 (Soto et al., 2003) (Caisson, Rexburgh, ID, USA) at

38.50C in an atmosphere 5% (v/v) CO2 in air.

Regardless of method of production, embryos greater than 16 cells in appearance

were collected at 1300 h on Day 6 or 7. Embryos were placed in groups of 21 to 65 into









2 ml cryogenic vials (Nalge Company, Rochester, NY, USA) filled to the top with

KSOM-BE2 that was pre-warmed and equilibrated in 5% (v/v) CO2 in air. Embryos

produced by Transova were kept separately from those produced using ovaries from the

local abattoir. Vials containing embryos were placed in a portable incubator (Minitube of

America, Verona, WI, USA) that had been pre-warmed to 390C for 24 h prior to use.

Embryos were shipped by air and arrived at Santa Cruz de la Sierra, Bolivia, at 1100 h

the next day (Day 7 or 8 after in vitro insemination) and transported by ground to the

farm.

Embryos were transferred over a time span from 1300 h and 2000 h. One or two

embryos were loaded into 0.25 cc straws in Hepes-TALP (Caisson) containing 10% (v/v)

bovine steer serum (Pel-Freez, Rogers, AR, USA) and 100 CLM 2-mercaptoethanol

(Sigma-Aldrich, St. Louis, MO, USA). Embryos were transferred to recipients that were

palpated the day before and had a detectable CL. Recipients were randomly assigned to

receive one or two embryos, and all embryos were transferred into the ipsilateral horn to

the CL. Pregnancy diagnosis was performed by rectal palpation at Day 64 and 127 post-

transfer, and the number of fetuses was recorded on Day 127. Data collected at calving

included length of gestation (with the day of transfer being considered Day 7 of

gestation), occurrence of dystocia (defined as needing assistance), sex, weight and

viability of each calf, and occurrence of retained placenta (failure of the placenta to be

expelled within 12 h after calving). Calf survival until Day 7 of age was also recorded.

Experiment 2 Administration of GnRH on Day 11 after Anticipated Ovulation in
Lactating Recipients that Received an IVP Embryo

This study took place at a commercial dairy located in Bell, FL, USA (290 45' N

82o 51' W) from June to October, 2004. Data on minimum and maximum air









temperatures and average relative humidity collected by the Florida Automated Weather

Service (http:.//fawn.ifas.ufl_ edu) for Alachua, FL, USA are presented in Figure 1. A total

of 87 multiparous, lactating Holstein cows in late lactation were used as recipients. Cows

were fed a total mixed ration to meet or exceed requirements recommended for lactating

dairy cows, milked three times a day, and received bovine somatotropin (Posilac",

500 mg sometribove zinc, Monsanto, St. Louis, MO, USA) according to manufacturer' s

directions. Cows were housed in a dry lot with access to a permanent shade structure

without fans or sprinklers and with access to a cooling pond.

Cows were prepared for embryo transfer in groups of 6 to 18; a total of 10

replicates were completed. To synchronize recipients for timed embryo transfer, cows

received 100 Cpg (i.m.) of GnRH (2 ml of Cystorelin ; Merial Limited, Iselin, NJ, USA),

on Day -10; 25 mg (i.m.) of PGF2,, On Day -3; and 100 Cpg (i.m.) of GnRH, on Day 0

(i.e., the day of anticipated ovulation). On Day 7 after anticipated ovulation, all cows

were palpated per rectum for the presence of a CL. Cows that had a palpable CL received

an epidural block of 5 ml of lidocaine (2%, w/v), and a single embryo was transferred to

the uterine horn ipsilateral to the ovary containing the CL. Recipients were randomly

assigned to receive 100 Cpg (i.m) of GnRH or vehicle (9 mg/ml of benzyl alcohol and 7.47

mg/ml of sodium chloride in water) on Day 11 after anticipated ovulation.

The embryos used for transfer were produced in the Gainesville laboratory using

oocytes of various breeds and a pool of semen from three bulls of various breeds as

described for Experiment 1. A different pool of semen was used for each replicate.

Presumptive zygotes were cultured in groups of ~30 in 50 Cll microdrops of modified

SOF (Fischer-Brown et al., 2002) containing 100 ng/ml of insulin-like growth factor-1









(Upstate Biotechnology, Lake Placid, NY, USA). Embryos were cultured at 38.50C in a

humidified atmosphere of 5% (v/v) 02 and 5% (v/v) CO2 with the balance N2. On Day 7

after insemination, blastocysts were harvested and transported to the farm in 2 ml

cryogenic vials (20 to 25 embryos/tube) filled to the top with pre-warmed Hepes-TALP.

Tubes containing embryos were placed in a portable incubator (Minitube of America,

Verona, WI, USA) that had been pre-warmed to 390C for 24 h prior to use. Embryos

were transported to the farm and loaded in 0.25 cc straws prior to transfer into recipients.

Pregnancy was diagnosed by rectal palpation at Day 45 to 53 after anticipated ovulation.

Statistical Analysis

Categorical data were analyzed by logistic regression using the LOGISTIC

procedure of SAS for Windows (Version 9, SAS Institute Inc., Cary, NC, USA) with a

backward stepwise logistic model. Variables were continuously removed from the model

by the Wald statistic criterion if the significance was greater than 0.2. The full statistical

model for Experiment 1 included treatment (one embryo or two embryos), parity (cows

vs heifers), estrus (observed in estrus vs not observed) and treatment x parity on

pregnancy rate, pregnancy loss, calving rate, calf mortality and twinning rate. The only

variable in the final mathematical model for Experiment 2 was GnRH treatment as other

effects (replicate and replicate x treatment) were not significant. The adjusted odds ratio

estimates and the 95% Wald confidence intervals (CI) from logistic regression were

obtained for each variable that remained in the final statistical model following the

backward elimination. Data were also analyzed with the GENMOD procedure of SAS to

determine the significance of each effect that remained in the reduced model; P values for

logistic regression analyses reported in the tables are derived from these analyses. Data

for gestation length and calf birth weight were analyzed by analysis of variance using









Proc GLM. The full statistical model included the effects of treatment, parity and

treatment x parity. The X2 test was used to determine whether the sex ratio of calves

differed from the expected 1:1 ratio.

Results

Experiment 1 Single or twin transfer of IVP embryos

Pregnancy and calving rates

Data are summarized in Table 1. At Day 64 of gestation, the pregnancy rate tended

to be higher (P=0.07) for cows than for heifers. While there were no significant effects

of number of embryos transferred or parity x number transferred, heifers that received

two embryos tended to have lower pregnancy rates than those that received a single

embryo (20% for two embryos vs 41% for one embryo) while there was no difference in

pregnancy rate due to number of embryos transferred to cows (50% for two embryos vs

57% for one embryo).

Pregnancy losses between Day 64 and 127 occurred in one group only cows

receiving two embryos. In that group, pregnancy rate was 50% at Day 64 but decreased

to 17% at Day 127. There was no difference in pregnancy rates at Day 127 between

cows and heifers, but recipients that received two embryos had lower pregnancy rates

(17% for cows and 20% for heifers) than recipients that received one embryo (57% for

cows and 41% for heifers, P < 0.03).

Pregnancy loss after Day 127 occurred in one female only. In particular, a cow

receiving a single embryo gave birth to a stillborn calf at 251 d of gestation. Like for

pregnancy rate at Day 127, there was no difference in calving rate between cows and

heifers, but recipients that received two embryos had lower calving rates (17% for cows









and. 20% for heifers) than recipients that received one embryo (50% for cows and 41%

for heifers, P < 0.03).

Estrus was detected at 24, 48 or 72 h after prostaglandin inj section in 21/32 heifers

(8 at 24 h after injection and 13 at 48 h) and 19/26 cows (1 at 24 h after injection, 14 at

48 h and 4 at 72 h). While not statistically different (P=0. 11), there was a tendency for

pregnancy rates to be lower for animals not detected in estrus. For example, pregnancy

rates at Day 127 for animals receiving one embryo was 55% (11/20) for animals in estrus

vs 36% (4/11) for animals not observed in estrus. Pregnancy rates at Day 127 for animals

receiving two embryos were 25% (5/20) for animals in estrus vs 0% (0/7) for animals not

observed in estrus.

Characteristics of gestation, parturition, and calves

Gestation length was affected by recipient type x number of embryos transferred

(P<0.05; Table 2). For cows, gestation length was slightly longer for those receiving one

embryo as compared to those receiving two embryos while the opposite was true for

heifers. Two of 5 females calving that received two embryos produced twin calves. There

was no significant effect of recipient type or number of embryos transferred on dystocia

or incidence of retained placenta (Table 2). Sex ratio (including the one stillborn calf)

was in favor of males with 15 males compared to 7 female calves born (68% male; Table

3). This ratio tended to be different from the expected 1:1 ratio (P<0. 10).

While there were no significant differences, there was a tendency for calf mortality

at birth to be greater for heifers receiving two embryos than for other groups (Table 3).

None of the cows lost their calf at birth and only 1 of 7 heifers receiving a single embryo

experienced calf death at birth. In contrast, 2 of 3 heifers receiving two embryos

experienced calf loss. One heifer had twin fetuses and both were born dead as a result of










complications with calving. Another heifer gave birth to a single calf that was born dead

as a result of complications with calving. The calf from the third heifer was born alive.

All calves born alive were alive 7 d later.

Experiment 2 Administration of GnRH on Day 11 after Anticipated Ovulation

Administration of GnRH at Day 11 after anticipated ovulation had no effect

(P>0.10) on pregnancy rates. Recipients treated with GnRH had a pregnancy rate of

17.8% (8/45) while those recipients that received placebo had a pregnancy rate of 16.7%

(7/42). The odds ratio was 1.08 with 95% Wald confidence interval of 0.23 and 3.30.

Discussion

The purpose of the experiments described here was to examine two strategies for

increasing pregnancy rates in heat-stressed dairy recipients that receive an IVP embryo.

Neither approach, transferring two embryos into the uterine horn ipsilateral to the CL or

inj section of GnRH at Day 11 after anticipated ovulation, increased pregnancy rates.

Results of Experiment 1 indicated that the transfer of two embryos into recipients

led to pregnancy loss and that such loss occurred earlier for heifers than for cows. There

was a distinct difference in pregnancy rate between heifers that received one or two

embryos as early as Day 64 of gestation. Among cows, in contrast, there were no

differences in pregnancy rate at this stage of gestation between recipients that received

one or two embryos. By Day 127, however, cows that received two embryos experienced

substantial mid-to-late fetal loss and pregnancy rate and subsequent calving rate was

lower for this group than for cows that received a single embryo.

The most likely explanation for the increased frequency of pregnancy loss in

recipients receiving two embryos is uterine crowding, with the effects of crowding

occurring sooner in gestation for nulliparous animals than for multiparous animals.









Similar results were obtained in another study (Anderson et al., 1979). In that study,

calving rates and twinning rates were similar for cow recipients regardless of whether

twin transfers were performed via bilateral or unilateral placement. For heifers, in

contrast, calving rate and twinning rate was lower for unilateral twin transfers than for

bilateral transfers. Using heifers, Rowson et al. (1971) also found lower embryonic

survival rates and twinning rates for recipients of unilateral twin transfers than for

recipients of bilateral transfers.

It is evident, however, that uterine capacity can vary between herds of cattle. Thus,

there were no differences in pregnancy success between recipients of twin embryos

placed unilaterally or bilaterally for heifers (Sreenan and Diskin 1989; Reichenbach et al.,

1992) or cows (Sreenan and Diskin 1989). Similarly, embryonic survival rate for beef

cows selected for twinning was similar for those having unilateral or bilateral multiple

ovulations (Echternkamp et al., 1990). In lactating dairy cows, in contrast, the likelihood

of a twin pregnancy resulting from multiple ovulation going to term was higher if

ovulations occurred bilaterally than if unilateral ovulations occurred (L6pez-Gatius et al.,

2005b). Perhaps, identification of the biological processes controlling uterine capacity

will lead to new approaches for increasing the efficacy of producing twins in cattle.

In an earlier study, administration of GnRH at Day 11 after anticipated ovulation

tended to increase pregnancy and calving rates in lactating Holstein recipients (Block et

al., 2003). The management of these cows was similar to those in Experiment 2. In both

studies, recipients were exposed to heat stress and received an IVP embryo using a timed

embryo transfer protocol. Effectiveness of treatment with GnRH or its analogues at 1 1

tol2 d after estrus for inseminated cows has yielded variable results, as some reports









indicated a positive effect (Sheldon and Dobson, 1993; Willard et al., 2003) while others

indicated no effect (Ryan et al., 1994). One factor that could influence the effectiveness

of GnRH treatment at Day 11 is the number of follicular waves that a female experiences

during an estrous cycle. Females with estrous cycles characterized by three follicular

waves have larger second-wave dominant follicles at Day 11 than females with two-wave

cycles (Ginther et al., 1989; Savio et al., 1990; Ko et al., 1991). Given that a follicle must

reach 10 mm in diameter to ovulate in response to LH (Sartori et al., 2001), the

preponderance of cycle type (two-wave vs three-wave) within a herd may determine

effectiveness of GnRH treatment at Day 11. Finally, it remains possible that failure to

observe an effect of GnRH treatment was because the number of animals per group was

low. The pitfalls associated with interpretation of experiments with low numbers has

been discussed (Amann, 2005) and could be responsible for the variation in results for

trials to test effects of GnRH on pregnancy rates in embryo transfer recipients.

Estrus is difficult to detect in lactating dairy cows because of the short duration of

estrus and the large proportion of cows that do not display intense mounting activity

(Dransfield et al., 1998). This problem, which is exacerbated by heat stress (Thatcher et

al., 1986), makes embryo transfer in lactating cows inefficient if recipient selection is

based solely on estrus detection. The first report of a timed embryo transfer protocol,

where ovulation was synchronized using an OvSynch protocol, was by Ambrose et al.

(1999). The suitability of timed embryo transfer as a method for preparing recipients was

demonstrated in Experiment 1 because calving rates were 50 and 41% for cow and heifer

recipients that received a single embryo, respectively. Similarly, using beef recipients, a

pregnancy rate of 49% was achieved using timed embryo transfer (Bo et al., 2002). In










contrast, pregnancy rate at Day 45 of gestation in Experiment 2 was only 17%. Low

pregnancy rates have been reported in other studies with timed embryo transfer using

lactating, heat-stressed recipients with pregnancy rates at ~ 45 d of gestation following

timed embryo transfer ranging from 11 26% (Ambrose et al., 1999; Al-Katanani et al.,

2002a; Block et al., 2003). The reason for the differences in pregnancy rates between

Experiment 1 and 2 cannot be deduced because of the large number of variables between

studies including nutrition, housing, level of milk yield, stage of lactation, breed,

synchronization protocol, and embryo culture protocol.

Despite the effectiveness of timed embryo transfer, there was a tendency for

pregnancy rates in Experiment 1 to be higher for those recipients detected in estrus. Most

of the animals not detected in estrus likely ovulated after the last GnRH inj section because

embryos were only transferred to recipients with a detectable CL. Nonetheless, some

cows in this group probably were not synchronized with respect to predicted ovulation

time.

Transfer of IVP embryos has been associated with large calf syndrome, increased

rates of fetal loss, sex ratio skewed towards the male and increased rate of dystocia and

calf mortality (see Hasler et al., 2000; Hansen and Block, 2004; Farin et al., 2004 for

review). There are also reports of prolonged gestation length (Kruip and den Dass, 1997;

Rerat et al., 2005). In Experiment 1, most characteristics of the fetus and calf that were

measured in females receiving one embryo were within normal ranges including

gestation length, rates of fetal loss, calf birth weight, and calf survival at birth and within

the first 7 d of age. The incidence of dystocia among females receiving one calf was 21%

and it is difficult to determine whether this value is high because of the particular mating









combinations used (embryos of diverse genotypes transferred into females of several

different genotypes). In a study with Holsteins bred by artificial insemination, the

frequency of difficult births ranged from 6 to 18% (Dj emali et al., 1987).

The one abnormality identified was a skewed sex ratio with 68% of the calves

being male. While previous work suggests that the altered sex ratio among IVP embryos

is due to toxic effects of concentrations of glucose in excess of 1 mM on female embryos

(Kimura et al., 2005), the concentration of glucose in the medium used for culture here

(KSOM-BE2) contains only 0.2 mM glucose (Soto et al., 2003). Others have found a

tendency for male embryos to become blastocysts sooner in development when cultured

in KSOM than female embryos (Nedambale et al., 2004b). Differences in sex ratio have

been seen as early as between the eight-cell and morula stages of development (Block et

al., 2003). While it is possible that selection of most embryos for transport done on Day

6 after insemination exacerbated the skewed sex ratio, Block et al. (2003) reported that

64% of calves born as a result of transfer of IVP embryos cultured in modified KSOM

were male even though embryos were harvested for transfer on Day 8 after insemination.

In conclusion, results indicate that unilateral transfer of two embryos to increase

pregnancy rate is unwarranted. The fact that fetal loss occurred sooner for heifers than

cows points out the importance of uterine capacity as a limiting factor for maintenance of

fetal development of two conceptuses. There was also no evidence that GnRH treatment

at Day 11 after anticipated ovulation improves pregnancy rate. Finally, the suitability of

timed embryo transfer as a method for preparing recipients for transfer was evident by the

high pregnancy and calving rates achieved with crossbred females that received a single

embryo. Additional research is warranted to reduce incidence of skewed sex ratio.









While sexed semen could be used to control sex ratio (Wilson et al., 2005), it is likely

that the underlying biological causes of altered sex ratio affect other aspects of embryo

physiology also.












Table 3-1. Effect of recipient type and number of embryos transferred per recipient on pregnancy rates and losses.
Pregnancy loss Pregnancy loss
Pregnancy rate, d Pregnancy rate, d
Reipen tpe64ofesatinb between Day 64 and between Day 127
127fgetaton" 127 of gestation Calving rate" and calving
Latain cw8/14 (57%) 8/14 (57%) 0/8 (0%) 7/14 (50%)1/(3%
single embryo
Lactating cow -
6/12 (50%) 2/12 (17%) 4/6 (66%) 2/12 (17%) 0/2 (0%)
two embryos
Nulliparous heifer -
7/17 (41%) 7/17 (41%) 0/7 (0%) 7/17 (41%) 0/7 (0%)
single embryo
Nulipros eier 3/15 (20%) 3/15 (20%) 0/3 (0%) 3/15 (20%) 0/3 (0%)>
two embryos
a Data are the proportion of animals pregnant of those that received embryos and, in parentheses, the percent pregnant.
b Logistic regression indicated effect of recipient type (P=0.07). The odds ratio estimate was 0.38 (heifer/cow) (95% Wald CI = 0. 13,
1.14; Wald Chi-Square statistic = 2.96, P=0.08).
" Logistic regression indicated an effect of number of embryos transferred (P<0.03). The odds ratio estimate was 4. 13 (one
embryo/two embryos) with a 95% Wald CI of 1.243, 13.690. Wald Chi-Square statistic 5.36; P<0.03).
d Data are the proportion of pregnant recipients at Day 64 that lost their pregnancy by Day 127 of gestation and, in parentheses, the
percent pregnancy loss.
e Data are the proportion of animals that calved of those that received embryos and, in parentheses, the percent pregnant.
* Logistic regression indicated an effect of number of embryos transferred (P<0.03). The odds ratio estimate was 3.62 (one
embryo/two embryos) with a 95% Wald CI of 1.090, 12.047. Wald Chi-Square statistic 4.41; P<0.04).
g Data are the proportion of pregnant recipients at Day 127 that lost their pregnancy before calving and, in parentheses, the percent
pregnancy loss.
h One cow expelled a stillborn calf at 251 d of gestation.









Table 3-2. Effect of recipient type and number of embryos transferred per recipient on
characteristics of pregnancy and parturition.
Gestation Twin Retained
Recipient type length, da pregnancieSb Dystociac placentad


)

)

)

)


Lactating cow -
282 + 3 0/7 (0%) 2/7 (29%) 4/7 (57%
single embryo
Lactating cow -
274 + 5 1/2 (50%) 0/2 (0%) 1/2 (50%
two embryos
Nulliparous heifer -
276 + 3 0/7 (0%) 1/7 (14%) 5/7 (71%
single embryo
Nulipfos eier 284 + 4 1/3 (33%) 1/3 (33%) 2/3 (67%
two emb~ryos-
aData are least-squares means + SEM. Gestation length was affected by recipient type x
number of embryos transferred (P<0.05).
b Data are the proportion of pregnancies in which twin calves were born and, in
parentheses, the percent pregnant. Logistic regression indicated an effect of number of
embryos transferred (P<0.02).
" Data are the proportion of pregnancies in which dystocia was recorded at birth and, in
parentheses, the percent cows experiencing dystocia.
d Data are the proportion of cows calving that experienced retained placenta and, in
parentheses, the percent cows experiencing retained placenta.









Table 3-3. Effect of recipient type and number of embryos transferred per recipient on
characteristics of calves born.
Sex ratio Calf birth Calf mortality
aMF) -egt kb Calf mortality
Recipient type at birth" today7
aged
Lacatig cw -5:3e 34 + 3 0/7 (0%) 0/7 (0%)
single embryo
Lactating cow -
2:1 25 + 5 0/3 (0%) 0/3 (0%)
two embryos
Nulipaoushei4: 3 26 +3 1/7 (14%)' 0/6 (0%)
single embryo
Nullipru heifer -
~ ~p~~s~~4:0 25 + 3/4 (75%)' 0/1 (0%)
a The overall sex ratio of 15 male and 7 females tended to be different (P<0. 10) than the
expected 1:1 ratio.
b Data are least-s uares means + SEM.
" Data are the proportion of calves that were bomn dead and, in parentheses, the percent bomn
dead.
d Data are the proportion of calves born alive that died before d 7 of live and, in parentheses,
the percent death before Day 7.
e Data includes the stillborn calf at 251 d of gestation
'One calf was stillborn from a cow not experiencing dystocia.
g One heifer had twin fetuses and both were born dead as a result of complications with
calving. The other two heifers gave birth to a single calf. One calf was born alive and the
other was born dead as a result of complications with calving.








71





Experiment 1- Bolivia


30



E 20-

S15-

10
Nov 1 Nov 15 Dec1 Decl5 Dec 30


100
90
80
[Y 70
60
Experiment 2 Florida s
35-





iEi



Q 10-


June 1 July1 Aug 1 Sept1 Oct1 Nov1


Figure 3-1. Maximum (open circles) and minimum (closed circles) daily air temperatures
and relative humidities (RH) during the experiments.















CHAPTER 4
EFFECTS OF HYALURONIC ACID IN CULTURE AND CYTOCHALASIN B
TREATMENT BEFORE FREEZING ON SURVIVAL OF CRYOPRESERVED
BOVINE EMBRYOS PRODUCED IN VITRO

Introduction

In vitro production of embryos is an important tool for improving genetic merit and

fertility of cattle and is an indispensable component of other technologies such as somatic

cell cloning and transgenesis (Hansen and Block, 2004). One limitation to the

widespread use of in vitro produced embryos in the cattle industry is the poor

survivability of in vitro produced embryos to cryopreservation. In vitro survival rates

following thawing (Pollard and Leibo, 1993; Enright et al., 2000; Khurana and Niemann,

2000a; Diez et al., 2001; Guyader-Joly et al., 1999) and pregnancy rates following

thawing and transfer (Hasler et al., 1995; Agca et al., 1998; Ambrose et al., 1999; Al-

Katanani et al., 2002a) are consistently lower for embryos produced in vitro when

compared to embryos produced in vivo by superovulation.

The poor survival of the in vitro produced embryo is associated with culture-

induced changes in ultrastructure (Rizos et al., 2002), gene expression (Bertolini et al.,

2002a; Lazzari et al., 2002; Lonergan et al., 2003), and metabolism (Krisher et al., 1999;

Khurana and Niemann, 2000b) that make it distinct from the embryo produced in vivo.

Among the metabolic changes are an increase in lipid content (Abe et al., 1999; Rizos et

al., 2002) and this condition has been linked to poor freezability. Mechanical delipidation

(Tominaga et al., 2000; Diez et al., 2001) and addition of inhibitors of fatty acid synthesis

(De la Torre-Sanchez et al., 2005) can improve survival following cryopreservation.









In the current study, two approaches for enhancing survival of bovine embryos

following cryopreservation were evaluated. The first was to culture embryos in the

presence of hyaluronic acid. This unsulphated glycosaminoglycan is present in follicular,

oviductal and uterine fluids in several species including cattle (Lee and Ax, 1984).

Receptors for hyaluronic acid (CD44) have been reported on the bovine oocyte, cumulus

cell, and preimplantation stage embryo (Valcarcel et al., 1999). Addition of hyaluronic

acid to culture medium has been reported to increase blastocyst re-expansion and

hatching after freezing (Stojkovic et al., 2002; Lane et al., 2003). The second approach

was to determine whether altering the cytoskeleton before cryopreservation would

enhance embryo survival. The rationale for this treatment is that cryoinjuries such as

intracellular ice formation and osmotic shock induce irreversible disruption in

microtubules and microfilaments (Kuwayama et al., 1994; Fair et al., 2001) and that

temporary depolymerization of actin microfilaments before cryopreservation could

reduce cytoskeletal damage and plasma membrane fracture caused by alterations in

cytoskeletal architecture (Dobrinsky, 1996). Addition of cytochalasin B to cause actin

depolymerization had no effect on survival of eight-cell embryos in the mouse (Prather

and First, 1986) but enhanced survival of expanded and hatched blastocysts without

effecting survival of morula and early blastocysts in the pig (Dobrinsky et al., 2000).

Materials and Methods

Embryo Production

Procedures, reagents, and media formulation for oocyte maturation, fertilization,

and embryo culture were as previously described (Roth and Hansen, 2005) with some

modifications. Briefly, cumulus oocyte complexes (COCs) were harvested from ovaries

of a variety of breeds collected at a local abattoir located at a travel distance of









approximately 1.5 h from the laboratory. The COCs were matured in Tissue Culture

Medium-199 with Earle's salts supplemented with 10% (v/v) steer serum, 2 Cpg/mL

estradiol 17-P, 20 Cpg/ml follicle stimulating hormone, 22 Cpg/ml sodium pyruvate, 50

Cpg/ml gentamicin and an additional 1 mM glutamine for approximately 22 h at 38.50C in

an atmosphere of 5% (v/v) CO2 in humidified air. Insemination with a cocktail of

Percoll-purified spermatozoa from three different bulls was performed in In Vitro

Fertilization Tyrode's Albumin Lactate solution. At 8 12 h post-insemination (hpi),

putative zygotes were denuded of cumulus cells by suspension in Hepes-TALP medium

containing 1000 units/ml hyaluronidase type IV (Sigma, St Louis, MO, USA) and

vortexing in a microcentrifuge tube for 5 min. Presumptive zygotes were then placed in

groups of ~30 in 50 Cll microdrops of a modified Synthetic Oviductal Fluid (SOF)

prepared as described by Fisher-Brown et al. (2002). Embryos were cultured at 38.50C in

a humidified atmosphere of 5% (v/v) CO2, 5% 02, and with the balance N2. Blastocysts

were collected for cryopreservation on day 7 after insemination.

Experimental Design and Embryo Manipulation

The experiment was a 2 x 2 factorial design to test main effects of hyaluronic acid

during culture (+ or -) and cytochalasin B before cryopreservation (+ or -). Data on

development were obtained from 18 replicates using 5022 oocytes while data on

cryopreservation were obtained from 7 replicates using a total of 197 blastocysts.

Following insemination and transfer to fresh microdrops, embryos cultured without

hyaluronic acid were cultured in SOF for 7 days beginning after insemination. Embryos

treated with hyaluronic acid were cultured in SOF until day 5 when all embryos were

transferred to a fresh microdrop of SOF containing 6 mg/ml hyaluronic acid from

Streptococcus zooepidemicus (Sigma).









Blastocysts and expanded blastocysts were harvested on the morning of day 7 after

insemination and washed twice in holding medium consisting of Hepes-TALP (Parrish et

al., 1989) containing 10% (v/v) fetal calf serum (FCS). Embryos treated with

cytochalasin B were incubated for 10 min at 38.50C in air while in Hepes-TALP

containing 10% (v/v) FBS and 7.5 Cpg/ml cytochalasin B (Sigma) in a 1.5 ml

microcentrifuge tube (Tominaga et al., 2000). Cytochalasin B was initially dissolved in

DMSO at a concentration of 5 mg/ml and was then added to HEPES-TALP to achieve a

final concentration of 7.5 Cpg/ml. Control embryos were incubated similarly in HEPES-

TALP containing 10% (v/v) FBS.

Cryopreservation

Procedures for freezing were modified from those reported elsewhere (Hasler et al.,

1995; Enright et al., 2000). In brief, blastocysts were transferred in groups of 10 to a fresh

100 Cl~ microdrop of Hepes-TALP containing 10% FCS at 3 8.5oC for the time it took to

harvest all embryos (~ 10 min). Next, embryos in groups of 5 8 per treatment

hyaluronicc acid or control) were randomly selected to receive cytochalasin B treatment

before freezing or not as described above. Afterwards, each group of 5 8 embryos was

placed in a 50 Cl~ microdrop of 10% (v/v) glycerol in Dulbecco's phosphate-buffered

saline (DPBS) containing 0.4% (w/v) bovine serum albumin (freezing medium) in a grid

plate over a slide warmer at 30oC. Within 10 min, embryos were loaded in a 50 Cl1 volume

into 0.25 ml plastic straws (Agtech, Manhattan, KS). Up to 8 embryos were loaded in

each straw. Two columns of 50 Cl1 freezing medium separated by air bubbles were always

placed above and below the column of embryos. Straws were transferred to a freezing

chamber (Cryologic Model CL5500 (Mulgrave, Victoria, Australia) for 2 min at -5oC and

then ice crystals were induced by touching the straw where the top column of medium









resided with a cotton plug that had been immersed in liquid nitrogen. After an additional

3 min at -5oC, embryos were cooled to -32oC at a rate of -0.6oC/min. After 2 min at -

32oC, straws were directly immersed in liquid N2 and stored until thawing (4 days 1

week later).

Thawing and Determination of Survival

Straws containing embryos were thawed by warming for 10 sec in air at room

temperature and 20 sec in a 32oC water bath. All subsequent steps before culture were

performed with media prewarmed to ~30oC and with dishes placed on a slide warmer set

at 30oC. Embryos were then expelled into an empty petri dish and immediately

transferred to a fresh 60 Cl1 drop of DPB S containing 6.6% (v/v) glycerol and 0.3 M

sucrose in an grid dish. After 5 min, embryos were sequentially transferred to DPBS

containing 3.3% (v/v) glycerol and 0.3 M sucrose for 5 min and DPBS + 0.3 M sucrose

for 5 min. Embryos were then washed three times in HEPES-TALP + 10 % (v/v) FCS

and placed into culture in groups of 5- 8 in 25 Cl1 microdrops of SOF containing 10%

(v/v) FCS. Culture was at 38.50C in a humidified atmosphere of 5% (v/v) CO2, 5% 02,

and 90% N2. Re-expansion was determined at 48 h after thawing and hatching at 72 h.

Statistical Analysis

The proportion of oocytes that cleaved and the proportion of embryos that

developed to the blastocyst stage on day 7 and day 8 were determined for each replicate.

Treatment effects were determined by least-squares analysis of variance using the proc

GLM procedure of SAS (SAS for Windows 90, Cary, NC). The model included the main

effects of replicate and treatment. Data for the proportion of frozen/thawed embryos that

re-expanded and on the proportion that hatched by 72 h of culture were analyzed using

the CATMOD procedure of SAS. The initial model included all main effects and two-









way interactions. After removing nonsignificant effects, the final model included

replicate, hyaluronic acid, preparation prior to freezing (none, cytochalasin B), and the

interaction of hyaluronic acid and preparation before freezing.

Results

Effect of Hyaluronic Acid on Embryonic Development

As shown in Table 1, addition of hyaluronic acid at day 5 after insemination caused

a slight reduction in the yield of blastocysts on day 7 and day 8 after insemination

regardless of whether data were expressed as the proportion of oocytes developing to the

blastocyst stage (P < 0.05) or the proportion of cleaved embryos developing to the

blastocyst stage (P < 0.01). Of the blastocysts that were recovered, 62-68% were

recovered at day 7 and the balance at day 8. There was no effect of hyaluronic acid on

the proportion of blastocysts collected at day 7 (Table 4-1).

Survival after Cryopreservation

Overall, cytochalasin B increased the percent of embryos that re-expanded

following thawing (P < 0.0001) and that hatched following thawing (P < 0.05) (Table 4-

2). Re-expansion rates were 51.2% (22/43) for embryos treated with cytochalasin B and

18.2% (8/44) for embryos not subjected to cytochalasin B. Hatching rates were 39.5%

(17/43) for embryos treated with cytochalasin B and 4.5% (2/44) for embryos not

subj ected to cytochalasin B.

While there was no significant effect of hyaluronic acid on cryosurvival, there was

a tendency (P=0.09) for a hyaluronic acid x cytochalasin B interaction affecting percent

of blastocysts that hatched following thawing. This interaction reflects the fact that

hyaluronic acid increased the percent hatching for embryos not subj ected to cytochalasin

B treatment and decreased percent hatched for embryos subj ected to cytochalasin B.









Discussion

Of the two treatments evaluated for enhancing cryosurvival of in vitro produced

bovine embryos, cytochalasin B treatment was the most effective as determined by an

improvement in both embryo re-expansion and hatching. The rationale for this treatment

is to reduce cellular injury caused by disruption in microtubules and microfilaments

(Kuwayama et al., 1994; Fair et al., 2001) and to increase flexibility of the plasma

membrane to allow it to tolerate forces associated with freezing that lead to membrane

damage. In other studies, addition of cytochalasin B had no effect on survival of eight-

cell embryos in the mouse (Prather and First, 1986), enhanced survival of expanded and

hatched pig blastocysts without effecting survival of morula and early blastocysts

(Dobrinsky et al., 2000), and improved survival of in vivo derived bovine blastocysts

subj ected to vitrification (Dobrinsky et al., 1995).

For embryos not exposed to cytochalasin B, there was a tendency for those cultured

in hyaluronic acid to have a higher re-expansion rate and hatching rate than embryos

cultured without hyaluronic acid. Both Stojkovic et al. (2002) and Lane et al. (2003)

reported improved survival rates to freezing when embryos were cultured in hyaluronic

acid; such a beneficial effect has not always been observed (Furnus et al., 1998).

Surprisingly, embryos cultured in hyaluronic acid were less likely to survive freezing

than control embryos when the cytochalasin B treatment was applied. Perhaps

physiological changes induced by hyaluronic acid cause the embryo to be less able to

adjust to the cellular actions of cytochalasin B. Those changes are potentially numerous

because hyaluronic acid acts to affect cell function through several means including

signaling through cell surface receptors, modifying the biophysical properties of

extracellular and pericellular matrices by attracting water, and by interacting physically









with a variety of ions and other molecules (Laurent, 1987; Ruoslahti and Yamaguchi,

1991; Hardingham and Fosang, 1992; Yasuda et al., 2002; Toole et al., 2005). One

possible mechanism by which hyaluronic acid could increase embryo survival to freezing

is by increasing the total number of cells in the embryo (Stojkovic et al., 2002; Jang et al.,

2003; Kim et al., 2005)

One unexpected finding was the reduction in the percentage of embryos that

became blastocysts caused by hyaluronic acid. In other studies, hyaluronic acid either had

no effect (Stojkovic et al., 2002; Lane et al., 2003) or caused an increase in blastocyst

yield (Furnus et al., 1998; Jang et al., 2003). Differences in origin and concentration of

hyaluronic acid could explain some of this difference between studies. Hyaluronic acid

can be isolated from different sources (ex., bacteria, rooster comb, and umbilical cord)

and preparations can differ in protein, endotoxin, and nucleotide content (Shiedlin et al.,

2004). Stojkovic et al. (2002) reported that preliminary results indicated that embryo

development in vitro was dependent upon the origin of the commercially-avail able

hyaluronic acid. However, embryos cultured with hyaluronic acid experienced a change

in culture medium at day 5 whereas control embryos did not. Such a difference could

have obscured beneficial effects of hyaluronic acid although another paper indicates no

effect of changing culture medium at 72 hpi on blastocyst yield in cattle (Ikeda et al.,

2000).

The percent of embryos that underwent hatching after freezing in glycerol and

thawing has varied from 0% (Enright et al., 2000) 22% (Diez et al., 2001; Nedambale et

al., 2004a), 32% (Guyader-Joly et al., 1999) and 69% (Hasler et al., 1997). The best

survival achieved in this study was for embryos cultured without hyaluronic acid and









treated with cytochalasin B. In this group, 51.2% of cryopreserved embryos were

capable of re-expansion and 39.5% hatched. It is likely that the percent hatching can be

further improved by modifying post-thaw culture-conditions. Massip et al. (1993) found

hatching rates for frozen/thawed, in vitro produced embryos were 41% when culture was

performed in the presence of bovine oviductal epithelial cells while hatching rate using

other culture conditions not involving co-culture was 0-6%. Nonetheless, one would not

expect optimal pregnancy rates to be achieved following direct transfer of embryos

frozen in glycerol even with the inclusion of cytochalasin B treatment. Rather, it is

suggested that pregnancy rates following transfer of embryos cryopreserved using slow-

freezing procedures can be optimized by selecting embryos for transfer based on

development in culture shortly after thawing.

In contrast to the poor survival of in vitro-produced embryos frozen using

conventional slow-freezing techniques, several experiments indicate that cryosurvival can

be enhanced by using vitrifieation (Vajta, 2000). It remains to be tested whether survival

of embryos produced in vitro after vitrifieation can be improved by cytochalasin B

treatment. There was a beneficial effect of cytochalasin B treatment on cryosurvival of

embryos derived in vivo following vitrifieation (Dobrinsky et al., 1995).

In conclusion, cytochalasin B treatment before freezing improved cryosurvival of

bovine embryos produced in vitro and subj ected to slow-freezing in glycerol. Such a

treatment could be incorporated into methods for cryopreservation of bovine embryos

provided post-transfer survival is adequate. In contrast, culture with hyaluronic acid was

of minimal benefit the increased cryosurvival in the absence of cytochalasin B was not

sufficient to allow an adequate number of embryos to survive.









Table 4-1. Effect of hyaluronic acid added at day 5 after insemination on production of
blastocysts at day 7 and 8 after inseminationab.
Culture Number Percent Blastocysts/oocyte Blastocysts/cleaved Percent of
medium of cleaved (%) c embryo (%) c total
oocytes blastocysts
that were
collected at
day 7
Control 1935 76.0 + 36.0 + 1.2* 47.2 + 1.3** 68.8 + 2.4
0.9
Haluronic 3087 77.7 + 31.5 + 1.2 40.7 + 1.3 62.2 + 2.4
acid 0.9

a n=18 replicates
b Means within a column that differ significantly are indicated by (P < 0.05) and ** (P < 0.01)
Includes blastocvsts collected at day 7 and those collected at day 8.


Table 4-2. Effect of culture in hyaluronic acid and treatment with cytochalasin B on
survival after cryopreservation. a
Cytochalasin
Culture medium treatment Re-expansion by 72 hb Hatching by 72 he

Control Control 8/44 (18.2%) 2/44 (4.5%)
Control Cytochalasin B 22/43 (51.2%) 17/43 (39.5%)
Hyaluronic acid Control 16/55 (29.0%) 7/55 (12.7%)
Hyaluronic acid Cytochalasin B 26/55 (47.3%) 12/55 (21.8%)
a Data are the fraction of embryos, and in parentheses, percent. Number of replicates was 7.
b Effect of cvtochalasin B (P < .0001).
Effect of cvtochalasin B (P < 0.05), hyaluronic acid (P < 0.10), and the cytochalasin B x
hyaluronic acid interaction (P = 0.09).















CHAPTER 5
GENERAL DISCUSSION

As alluded to at the beginning of this thesis, there has been a precipitous decline in

fertility of dairy cows over the last 10-40 years in North America (Butler, 1998), Ireland

(Roche, 2000), Spain (L6pez-Gatius et al., 2003), and the United Kingdom (Royal et al.,

2000). In addition, heat stress can compromise fertility in lactating dairy cows (Putney et

al., 1989b; Al-Katanani et al., 1999). The purpose of the present series of experiments

described in the thesis was to 1) evaluate strategies for enhancing fertility after AI using

GnRH treatment (Chapter 2) and 2) further develop ET using in vitro produced embryos

as a tool for increasing fertility by testing whether pregnancy rate could be improved by

transfer of twin embryos (Chapter 3) and whether the developmental competence of

embryos after cryopreservation could be improved by hyaluronan or cytochalasin B

treatment (Chapter 4). Results indicated no consistent benefit of injection of GnRH at

Day 11-15 after anticipated ovulation or insemination on pregnancy rates in heifers or

lactating cows. While unilateral transfer of two embryos was not shown to be an effective

treatment for increasing pregnancy rate in recipients, the high pregnancy rates achieved

in this study point to the potential usefulness of ET as a tool for enhancing fertility.

Large-scale use of embryo transfer will require the ability to freeze embryos successfully.

Results suggest that treatment of embryos with cytochalasin B before freezing is a

promising tool for enhancing survival of embryos following cryopreservation. A large

number of studies have been performed to test the effect of GnRH administration after

expected ovulation on fertility of cattle. Previous results indicated that GnRH was









sometimes effective at increasing pregnancy rate, but this beneficial effect was often not

observed (Peters et al., 2000). Despite this knowledge, we chose to reevaluate the

effectiveness of GnRH treatment because of a report that GnRH treatment at Day 11 after

estrus increases pregnancy rates in lactating cows exposed to heat stress (Willard et al.,

2003). Accordingly, it was hypothesized in Chapter 2 that the beneficial effect of GnRH

treatment would be greater during the summer than winter. This may be so because the

antiluteolytic process may be compromised by heat stress because of decreased growth of

the filamentous stage concepts (Biggers et al., 1987) and increased uterine PGF2a

secretion from the uterus (Wolfenson et al., 1993).

Overall, the results of GnRH treatment were generally negative. For treatment at

Day 11i, a positive effect of GnRH on fertility was never seen. This was the case for

heifers and lactating cows subj ected to AI or whether animals were exposed to heat stress

or not (Chapter 2; experiment 1 and 2). Treatment of lactating recipients with GnRH at

Day 11 also failed to increase pregnancy rate during heat stress in ET recipients (Chapter

3, experiment 2). Effectiveness of treatment with GnRH or its analogues at 11 to 12 d

after estrus for inseminated, heat-stressed lactating cows has yielded variable results, as

some reports indicated a positive effect (Sheldon et al., 1993; Willard et al., 2003), while

others indicated no effect (Jubb et al., 1990). Also, administration of GnRH at Day 1 1

after anticipated ovulation tended to increase pregnancy and calving rates in lactating

Holstein embryo transfer recipients exposed to heat stress (Block et al., 2003).

One factor that could influence the effectiveness of GnRH treatment at Day 11 is

the number of follicular waves that a female experiences during an estrous cycle. Females

with estrous cycles characterized by three follicular waves have larger second-wave









dominant follicles at Day 11 than females with two-wave cycles (Ginther et al., 1989;

Savio et al., 1990; Ko et al., 1991). Given that a follicle must reach 10 mm in diameter to

ovulate in response to LH (Sartori et al., 2001), the preponderance of cycle type (two-

wave vs three-wave) within a herd may determine effectiveness of GnRH treatment at

Day 11.

In one experiment (Chapter 2; experiment 3), administration of GnRH at Day 14

after anticipated ovulation in cows subj ected to TAI increased pregnancy rates of

lactating cows in the summer and winter at two locations. In the following year, though,

GnRH failed to improve fertility when treatment was administered either at day 14 in

cows subj ected to TAI (experiment 4) or at day 14 or 15 in cows previously diagnosed

coming in estrus (experiment 5). It is important to recognize that GnRH treatment should

improve fertility only when triggering luteinization or ovulation of developing

estrogenicc) follicles. Thus, there are at least two possible reasons for a lack in response

upon GnRH treatment at day 14 or 15. One possibility relates to the timing of ovulation

relative to the GnRH treatment and whether these animals failed to ovulate after being

diagnosed as coming into estrus. Although after observing estrus one does not expect

ovulation to fail, this expression does not necessarily mean that subsequent ovulation

occurred (L6pez-Gatius et al., 2005b) and insemination after a false identified estrus

often occurs (Heersche and Nebel 1994). According to L6pez-Gatius et al. (2005b), the

risk of cows failing to ovulate (12%) during the summer was greater than in the cool

period (3%).

During experiment 3 all cows received a GnRH inj section at 72 h following

PGF2a to insure an ovulation of the synchronized dominant follicle. Perhaps, the positive









GnRH effect observed during experiment 3 was masked in the following experiment

because cows did not receive an additional GnRH dose at estrus to ensure subsequent

ovulation. According to Lopez-Gatius et al. (2005a), there is evidence demonstrating the

benefits upon GnRH treatment when given on the day of insemination compared to

controls (30.8% vs. 20.6%), but conception rates were greater if cows received an

additional dose at day 12 post-insemination (35.4%). On the other hand, when GnRH

treatment took place on day 15 to ensure a responsive estrogenicc) dominant follicle

would ovulate at the time of GnRH treatment, it failed to improve fertility as well.

Similarly, in a recent study (Bartolome et al., 2005) there was no effect of GnRH

treatment on pregnancy rates of lactating cows when administered either on day 15 or day

5 and 15 after TAI.

It remains possible that inconsistency in effects of GnRH treatment is caused in

part by the low number of animals per treatment group. The pitfalls associated with

interpretation of experiments with low numbers has been discussed (Dransfield et al.,

1998) and could be responsible for the variation in results for trials to test effects of

GnRH on pregnancy rates in embryo transfer recipients and for inseminated cows.

With an existent variation among trials regarding the use of GnRH at day 1 1-15

post-insemination, one could speculate that such inconsistency regarding treatment is due

to the fact that herds of cattle determine the result that an experiment achieves. However,

our results indicate that such a hypothesis is not likely because when an experiment was

replicated the next year using the same herd, GnRH treatment once again proved to be

inconsistent in improving pregnancy rates.









According to Thatcher et al. (2005), hCG results in a more prolonged rise in LH

activity than is achieved following GnRH treatment. Perhaps the likelihood of ovulating

or luteinizing the dominant follicles present at the time of treatment would be higher

using hCG. Although low numbers of inseminated animals were used (n=8; n=49) hCG

treatment on d 14 after estrus improved pregnancy rates (Raj amahendran and

Sianangama, 1992; Sianangama and Rajamahendran, 1992). Use of hCG warrants further

investigation for any additional effect or response during the summer to enhance

pregnancy rates of lactating cows.

Recent work has focused on use of ET to bypass early embryonic death (Putney et

al., 1989b; Ambrose et al., 1999; Al-Katanani et al., 2002a). Given that ET can be more

effective at increasing pregnancy rates than AI for lactating cows during periods of heat

stress (Putney et al., 1989b; Ambrose et al., 1999; Drost et al., 1999; Al-Katanani et al.,

2002a), the potential benefit of ET can be realized. For ET to become an economical

alternative to AI on a wide scale basis in commercial herds, embryos must be inexpensive

to produce (Hansen and Block et al., 2004). Although embryos produced using IVP

systems are relatively inexpensive as compared to embryos produced by superovulation,

pregnancy rates achieved following transfer of an IVP embryo are often less than what is

obtained following transfer of an embryo produced by superovulation (Hasler et al.,

1995; Agca et al., 1998; Ambrose et al., 1999; Al-Katanani et al., 2002a). In addition,

IVP embryos are less likely to survive freezing than superovulated embryos (Hasler et al.,

2003), likely due to their increased lipid content (Abe et al., 1999; Rizos et al., 2002).

Accordingly, the second approach for the thesis focused on improvements in ET by










comparing pregnancy rates following the transfer of two embryos compared to one and

by increasing the viability of embryos that were cryopreserved.

The first effort was to determine whether transfer of two IVP embryos into the

uterine horn ipsilateral to the CL could increase pregnancy rates during periods of heat

stress. It was hypothesized that such a treatment might increase pregnancy rates because

the likelihood is increased that the cow receives at least one embryo competent for

sustained development. In addition, the transfer of two embryos into the ipsilateral

uterine horn is likely to increase the amounts of interferon-z and other embryonic

signaling molecules in the uterus needed to maintain pregnancy and prevent luteolysis.

Transferring two embryos into the uterine horn ipsilateral to the CL failed to

increase pregnancy rates. Instead, the transfer of two embryos into recipients led to

pregnancy loss, which occurred earlier for heifers than for cows. The most likely

explanation for the increased frequency of pregnancy loss in recipients receiving two

embryos is uterine crowding, with the effects of crowding occurring sooner in gestation

for nulliparous animals than for multiparous animals. Regardless of whether twin

transfers were performed via bilateral or unilateral placement, similar results were

obtained in another study (Anderson et al., 1979). In contrast, calving rate and twinning

rate in heifers was lower for unilateral twin transfers than for bilateral transfers.

Similarly, Rowson et al. (1971) also found lower embryonic survival rates and twinning

rates for recipients of unilateral twin transfers than for recipients of bilateral transfers in

heifers.

It is evident that uterine capacity can vary between herds of cattle. Thus, there were

no differences in pregnancy success between recipients of twin embryos placed









unilaterally or bilaterally for heifers (Sreenan et al., 1989, Reichenbach et al., 1992) or

cows (Sreenan et al., 1989). Similarly, embryonic survival rate for beef cows selected for

twinning was similar for those having unilateral or bilateral multiple ovulations

(Echternkamp et al., 1990). In lactating dairy cows, in contrast, the likelihood of a twin

pregnancy resulting from multiple ovulations going to term was higher if ovulations

occurred bilaterally than if unilateral ovulations occurred (L6pez-Gatius and Hunter,

2005). Perhaps, identification of the biological processes controlling uterine capacity

will lead to new approaches for increasing the efficacy of producing twins in cattle.

An additional limitation to the widespread use of IVP embryos in cattle is their

poor survival following cryopreservation. In vitro survival rates following thawing

(Pollard and Leibo, 1993; Enright et al., 2000; Khurana and Niemann, 2000a; Diez et al.,

2001; Guyader-Joly et al., 1999) and pregnancy rates following thawing and transfer

(Hasler et al., 1995; Agca et al., 1998; Ambrose et al., 1999; Al-Katanani et al., 2002a)

are consistently lower for IVP embryos when compared to embryos produced in vivo by

superovulation.

The percent of embryos that underwent hatching after freezing in glycerol and

thawing has varied from 0% (Enright et al., 2000), 22% (Diez et al., 2001; Nedambale et

al., 2004a), 32% (Guyader-Joly et al., 1999), and 69% (Hasler et al., 1997). Of the two

treatments evaluated for enhancing cryosurvival of IVP bovine embryos, cytochalasin B

treatment was the most effective as determined by an improvement in embryo re-

expansion and hatching rates. In this treatment, 51.2% of cryopreserved embryos were

capable of re-expansion and 39.5% hatched. Nonetheless, one would not expect optimal