<%BANNER%>

Effects of Dietary Aluminum from Water Treatment Residuals on Phosphorus Status and Bone Density in Growing Lambs

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 E20101123_AAAACZ INGEST_TIME 2010-11-23T16:04:01Z PACKAGE UFE0011627_00001
AGREEMENT_INFO ACCOUNT UF PROJECT UFDC
FILES
FILE SIZE 6567 DFID F20101123_AABTSK ORIGIN DEPOSITOR PATH vanalstyne_r_Page_38thm.jpg GLOBAL false PRESERVATION BIT MESSAGE_DIGEST ALGORITHM MD5
e83cb807c3208685e4dc8ac6a8bd1620
SHA-1
3929c76b06767b15b42208ffd48ae3c33656b1c5
1053954 F20101123_AABTIQ vanalstyne_r_Page_38.tif
981530080a91d4a5fd33f78f988c82a5
40b2717e3d38d68e0a98bb2d9a1f70da54896a2a
62854 F20101123_AABTDT vanalstyne_r_Page_56.jpg
c51277d2a298a3eebdd8d6d041753ebc
755ea6d52a62fc1b709be55dc7431d157217088c
2005 F20101123_AABTNN vanalstyne_r_Page_20.txt
e529889194fb58b79f12c8586a31d74b
90deedbfdd11883531e0c0eec36f5360d0772afd
24852 F20101123_AABTSL vanalstyne_r_Page_39.QC.jpg
4729470b20c60571ca2191ce4a2852f9
883c28df4cfeacaf525e91b9143b9dbfda154a2f
F20101123_AABTIR vanalstyne_r_Page_39.tif
05420cad7fcde2c3cecbe91312d67ce8
391481160916051a4580c4850fe66266a13a4f7b
66148 F20101123_AABTDU vanalstyne_r_Page_57.jpg
c89e2499b9b0b085afd0b4b145610ca0
05e1f4c62f89f97e22950ac0bb0388bfc63c41fc
1951 F20101123_AABTNO vanalstyne_r_Page_21.txt
ce4890c67ba10055997646e497f8f27a
877f70c8da90bf2562c185d44b7e5e60749e0981
6824 F20101123_AABTSM vanalstyne_r_Page_39thm.jpg
0a5a368bd0f52d43b98aff401cf7b8ca
5d798c5826bb6cb0dc4c1f3e1cd6a8f6b4f1cf0b
F20101123_AABTIS vanalstyne_r_Page_40.tif
beab48f9e05b1c3dcda69ba57d99576d
f12ec4c4fe5b1124dbfcbb8ec6143d0e2a27fa2d
74067 F20101123_AABTDV vanalstyne_r_Page_58.jpg
99039cc4faf31fd0cbcde0d3b3d7c806
bf3be79d69863d05956b7b6a377f1b6b01f7925b
1805 F20101123_AABTNP vanalstyne_r_Page_22.txt
17b12779a82447c8e3a55322cc4ce5fb
234c140a0e64ed13da5ce78dccb46bc0608d6d26
23709 F20101123_AABTSN vanalstyne_r_Page_40.QC.jpg
edff70b2047e830251e2f8d1693d9bcf
d8a8bb268d82c39e9c06f777409ad0baf8c9f7c4
1890 F20101123_AABTNQ vanalstyne_r_Page_23.txt
d6ee342e7354c02b5c6de808bdbea816
55d37a920dd11fdaa1750ffa5245a4ffa74614bc
F20101123_AABTIT vanalstyne_r_Page_42.tif
d55d3bf33ec66250f465ffc863014590
6f30330e47b9c1de525c566eb5b5d6576a03a155
6461 F20101123_AABTSO vanalstyne_r_Page_40thm.jpg
c7790f29c7ab780957bffb7b5d6522ab
cec94dc5aaa18bb89cc8f87b2c49856443a501bd
71486 F20101123_AABTDW vanalstyne_r_Page_59.jpg
26fd2bc8385b0feabd8edce41062db65
c3cb2f70705cf4153fdac8e82fb26d86fb0b4e2e
2047 F20101123_AABTNR vanalstyne_r_Page_24.txt
b21d4d9ed747419dd480a9d5aef1054c
5bfd6e65ec0fe4ad1234a72a09dd71725cbdca46
F20101123_AABTIU vanalstyne_r_Page_43.tif
fb72d21ac904b01e2b2998b687ae64f8
8aeb73bdf782ba257040d13dda66272fcc9a041c
24523 F20101123_AABTSP vanalstyne_r_Page_41.QC.jpg
07ae7a03642c1f4a4ccc55da49aed84b
bfb34ed079e38bce446cb8635176a8f7c4b9cfcf
27487 F20101123_AABTDX vanalstyne_r_Page_60.jpg
928d5dbe3356520539efccdca22ada8e
4cb7346cab1d5d192514aeb81555db770fe374c3
1957 F20101123_AABTNS vanalstyne_r_Page_25.txt
7175640840d0deb24e5d6f402ec6d591
58eba32e141d16cd6b3e2a236e8be784fe35907e
F20101123_AABTIV vanalstyne_r_Page_44.tif
61154df1de661aac978018d65b29a4dd
9fb310801323175359400ef0b2c4736df472227b
6702 F20101123_AABTSQ vanalstyne_r_Page_41thm.jpg
a95c61b8f74fd8f9e9fc86de7e02ceac
a1642be4b4a8df8acb1556e021087bbefacfbdf6
113795 F20101123_AABTBA vanalstyne_r_Page_58.jp2
c83d28eb4c6f97bab3483b14a4e96833
1a7e0986b09a9bb656bec701ca65a525f0a5eef0
80943 F20101123_AABTDY vanalstyne_r_Page_61.jpg
d22a82640270ec43bdf44bb895011aaa
1cac66605004c6f062db29442cca5c647f95dd14
2017 F20101123_AABTNT vanalstyne_r_Page_26.txt
c8df46dff802f4370f7c0e5f9444be1c
274d5843938b62d8d7ce76f86be81582cdf15c32
F20101123_AABTIW vanalstyne_r_Page_45.tif
f77397a9561ba4a4e279b7327af0b6d0
0aaeb66381a43873ef100493ac5aae57401d3be0
23890 F20101123_AABTSR vanalstyne_r_Page_42.QC.jpg
94c52cd6bd3ff7995fd5cc3169eb0f7e
27071d8cfdb15fa122733801ac5949c4bd1be915
6080 F20101123_AABTBB vanalstyne_r_Page_57thm.jpg
b622330b78701da2dac566ef158490f5
8faa7322983c8bff9dfed43aa50aadd75527c947
81168 F20101123_AABTDZ vanalstyne_r_Page_62.jpg
25e7fc25fbff7ad42d2942f9133024ef
92e747e90aa863c9a638292945306a70153d533e
1960 F20101123_AABTNU vanalstyne_r_Page_27.txt
4ec2ca3a7c24e64b43e7311b006aeb08
ddffb77c86234213bcd2e882c3e440706e633c76
F20101123_AABTIX vanalstyne_r_Page_46.tif
afe2ce3de2ab137a3da77c8ea4a194fb
dc677740c82dfffbadeafcb168e5c3ba300f6e73
6509 F20101123_AABTSS vanalstyne_r_Page_42thm.jpg
898bc3722b0215f6bc3886665de7e32d
45b7eaa2decdff30817e2aae654089ceb022fca4
47014 F20101123_AABTBC vanalstyne_r_Page_55.pro
110bc0749218bce9a1b5633112fd2cda
83f73198caf4ec1b7657c02041c28417f58b153c
2031 F20101123_AABTNV vanalstyne_r_Page_28.txt
4e33d7670c9a57e49c5e61e43f7d3e04
8d6a15b853baba44c1d22c3a23cb2cf202c9c43b
F20101123_AABTIY vanalstyne_r_Page_47.tif
ba8bcae953e1910f892f09ec112f31cf
058b70aa5dd3ef204124290c337ac92e7ed7c779
23972 F20101123_AABTST vanalstyne_r_Page_43.QC.jpg
e2c53e26f25dffe9af1dfb9b89e7a507
5f5e9e74c4624240535e94baba9558bb1deb7fba
6085 F20101123_AABTBD vanalstyne_r_Page_65thm.jpg
a55304d68bc3d002aa43b013c5a76170
ecea25ae7a62027eae4b9f4c69252aa1a12fc228
1996 F20101123_AABTNW vanalstyne_r_Page_29.txt
e687c2edd328fa4fc9d828c0ff4940aa
d2e09fa9c771ee4a783cd8893156e7856f4b029b
109194 F20101123_AABTGA vanalstyne_r_Page_42.jp2
7fba7ace2a481e580f2d4dedb1b581ff
c35d3313f03303c597f4fb54aac0e0d6bd3e3755
F20101123_AABTIZ vanalstyne_r_Page_48.tif
fcfbdb87642ec9fa16392e84586f0991
884ee8d6d609f49e6efa0802950427fe030a1dc0
6639 F20101123_AABTSU vanalstyne_r_Page_43thm.jpg
b8c70bad28c961700f0df65ee797547c
f2f85042bdad261ecd60454ce29d88432ae37817
6653 F20101123_AABTBE vanalstyne_r_Page_15thm.jpg
ece2c6b830e363ce267cbaa263a75b64
39a5b378a0fc31e62fbf6b59717ae322b61287ff
2012 F20101123_AABTNX vanalstyne_r_Page_30.txt
cad0990094113af89e6567fa4ff19a96
0ad7b3b2067b725c68b61eefda682bb98b6b3b6b
109872 F20101123_AABTGB vanalstyne_r_Page_43.jp2
f39f2fe075d7e9b705ea8ae10f948649
42cac5ce53c76dc361b86f7b683bec9d3242bdae
6474 F20101123_AABTSV vanalstyne_r_Page_44thm.jpg
ea1dd1eb79b926f86d372e6450fbc374
27fdd474e8f2f715e2fd2e12937d1c1e3a3258d1
3658 F20101123_AABTBF vanalstyne_r_Page_50thm.jpg
b31998257e841aec13f2ffe05a68504c
e0b7dbc7af01e39412126c862b7f7827e24b9ce5
2028 F20101123_AABTNY vanalstyne_r_Page_31.txt
751b0553a03fdfbc1cdab659bf67d9ba
4997a278047efee9d8e0b900a6dd7be3a8ca51ef
108411 F20101123_AABTGC vanalstyne_r_Page_44.jp2
189681f52f93a466969b3867a2939e1b
5949e1fc8ec6be8e0409a423fef263b1aaab4769
23717 F20101123_AABTSW vanalstyne_r_Page_45.QC.jpg
6fa999312ebfe4cb93548d0707ecb398
67fb7991123ee02916e0205df5fe9e32e6c2644c
49633 F20101123_AABTLA vanalstyne_r_Page_27.pro
b8b1231d9db326eda80612c152a77ac1
40756c97ce664b736be88a5f0514b72348827ea1
556 F20101123_AABTNZ vanalstyne_r_Page_32.txt
0f036e83e2bc7602287d6c79f29cdc96
7520e9c755c33942df17e2839cf9f0da287ac41e
110610 F20101123_AABTGD vanalstyne_r_Page_45.jp2
d0367546e7c579d19b72bf8ae4ed47ac
eb692707bf04529878260933b10ef64cf77cf6af
F20101123_AABTBG vanalstyne_r_Page_02.tif
35ccb702d5de30a36d384daa2ccc5fe1
a8d35e48c892b89a4c40dbf7cd9ecdb1e6e41997
6605 F20101123_AABTSX vanalstyne_r_Page_45thm.jpg
419881d320462e91f0572b2e375f56e3
2657a861f3da29fb12e002ae71da2f628b8769d5
51600 F20101123_AABTLB vanalstyne_r_Page_28.pro
03011c89d9cc1e77cd8d01ccc4f706e1
251797323c54a545076eb663871560c557fa3721
109062 F20101123_AABTGE vanalstyne_r_Page_46.jp2
44ce8ea5529f607a9eaf8a3ef6707dbe
f6bd02ae904cad14bca4994583f175820fbae27c
72334 F20101123_AABTBH vanalstyne_r_Page_44.jpg
06eccb43e78e8e1944364c842d41f15c
1ec97991c8b1a29b8b7faa44e752838255806c44
23897 F20101123_AABTSY vanalstyne_r_Page_46.QC.jpg
820b5e19591757a5ee1bd7adde7ea8ac
41a8c8096e282edda4a50874fd5c72da9cdef66e
49793 F20101123_AABTLC vanalstyne_r_Page_29.pro
7c6b23b055b66a7ab46ae89d1ed4a92d
f3c00b77744ce5b14b7743a10ed6e140d0d48cbe
54904 F20101123_AABTGF vanalstyne_r_Page_47.jp2
6e45b5e9c73c871356bb529003785830
39aa99e421e832e99ad42ec810a9e295bd114c23
2627 F20101123_AABTQA vanalstyne_r_Page_05thm.jpg
7931cf0248467f96996c5be778eb4302
cf11b1050cdb323c9bfb83ff36dd2a4adf3009a5
F20101123_AABTBI vanalstyne_r_Page_41.tif
929fcab13486b76a09c8917684a879e6
bc819aabec938a7fda3f93382c396b082a657286
6803 F20101123_AABTSZ vanalstyne_r_Page_46thm.jpg
43cf6d3ab4e0077c487cd756b77f2f33
73da0b568bbd0e961ed7141c01c231d1298d6ab2
51282 F20101123_AABTLD vanalstyne_r_Page_30.pro
584d8602a52dab13ec9df841d95e90a6
5bf4f2e07ee1389566108fbc14f15714c5bdf59b
122442 F20101123_AABTGG vanalstyne_r_Page_48.jp2
c6ffbfb4479dde38d184ba59bf3d0cef
fbf7d2c8ab1ae7f58c97d7cf9a3b4c7a1dc96654
16572 F20101123_AABTQB vanalstyne_r_Page_06.QC.jpg
49de3d7bc4e4887a29b61672e7d23c05
c453aa8343e3c7d79def4954708c4c9ae66c0bb4
8566 F20101123_AABTBJ vanalstyne_r_Page_32.QC.jpg
6eb3787f0b228cd0b83031975bb5d479
164b1b521527fe8e88e7ffee4b5c51dea1e8c35b
51461 F20101123_AABTLE vanalstyne_r_Page_31.pro
aa14b1a3869f9b62b7fab6b4185916ba
3e679001bf84875a15849a4c2694a8dc054c3978
113315 F20101123_AABTGH vanalstyne_r_Page_49.jp2
7315bc6d81991f0460f617cb188391de
a67e9e8f87f798475da1a2ac9768b340259fc3ca
4314 F20101123_AABTQC vanalstyne_r_Page_06thm.jpg
45064fc4ddd5a8c9f0be0f087f3d9c77
5e0fa2ed4ad66c45948c95b360a438cf080ee64e
21285 F20101123_AABTBK vanalstyne_r_Page_10.QC.jpg
805bbc2436deb13880734fe380b2673a
97aed9d0d0830f444eea0e30665c1f8bd0b2a0a5
88485 F20101123_AABTVA UFE0011627_00001.mets FULL
b57460bbd6a1d24a37841a499574d2bb
3351e9a32fad8e502e5933beab60b759142ffe72
12830 F20101123_AABTLF vanalstyne_r_Page_32.pro
0fcd3d0a44c72d7bf9925d197e0a6701
53756dd195d5184a9f9cadd24fe1db1481145cce
60445 F20101123_AABTGI vanalstyne_r_Page_50.jp2
e970349b9bc802cfd504e0bc445adeb8
dc29f5d6a85d49886e572617ebe23cebc1d84dc7
23647 F20101123_AABTBL vanalstyne_r_Page_44.QC.jpg
06445d66ee0e1c93fede85c845c9520f
2e024bbb6e450b3c34d1dc0321654166dc90f559
38433 F20101123_AABTLG vanalstyne_r_Page_33.pro
df5ebd693a811d52afce30b2aadf24a1
79262fd967ff2621cb2302cdec4fd8706b5c1a89
105497 F20101123_AABTGJ vanalstyne_r_Page_51.jp2
a419bedd3c68c276d6915528c1f2160d
21c2592a92e31ec8f3da8da9f2cf05fa03040de0
12944 F20101123_AABTQD vanalstyne_r_Page_07.QC.jpg
b94071158db57f23a42f768a42ce2cfa
049079aecc6f7cec545572a9b5c7c19c85fc0dc8
35968 F20101123_AABTBM vanalstyne_r_Page_66.pro
2fee52264b2fb3d79c84302ca57b13aa
77075e052d7d513e540eb613a7f977933b46bb51
48189 F20101123_AABTLH vanalstyne_r_Page_34.pro
4a38054889cb982b84d30d105c1c882e
6527918a68f694b15b9255ef1f2ce4a0a0e64d16
466153 F20101123_AABTGK vanalstyne_r_Page_52.jp2
82df0269fab0952fa9cd7550ab8c090f
eeca1dff7616d06602be093b7b8a928cbb019f34
3489 F20101123_AABTQE vanalstyne_r_Page_07thm.jpg
719d72a1780d7a5a806d928e504e787a
7c76f15299532adb97d817c0985e62878b762362
23220 F20101123_AABTBN vanalstyne_r_Page_36.QC.jpg
b605bd91982fe26bc66111b0afcf4fba
ea87d3a91bcdf4abcf8b2d01e76596db6068c63c
48260 F20101123_AABTLI vanalstyne_r_Page_35.pro
90676d93f23262671775fcf1bb2204fa
1e0db49773c88e563a2e61e3d0dfb554afd7dee5
92539 F20101123_AABTGL vanalstyne_r_Page_53.jp2
19b8a547ac018542582d144ac308ece1
8f5d3fd0a34b27a44c6bb5426bd5df74595c2d77
14141 F20101123_AABTQF vanalstyne_r_Page_08.QC.jpg
d61dc6547a7cc75e2a1040fb0fd7125b
22e3f58ce77056808aa80d0605034f21466eb9c6
114560 F20101123_AABTBO UFE0011627_00001.xml
00c6068fc0634bf5ac1cde18fb5fa03f
a90404176f552eb5eb40397fdda12e64853fdca4
48057 F20101123_AABTLJ vanalstyne_r_Page_36.pro
cb658d42a8751c697d30c8d69b5de274
6f3821105846ff9ce38c8323d043be4a2677fdae
110969 F20101123_AABTGM vanalstyne_r_Page_54.jp2
2da1bbc5f0b50c92a605dc5943886fbe
afe52d149e99b13a68797f76a09b37b01a73ffc3
4107 F20101123_AABTQG vanalstyne_r_Page_08thm.jpg
67d13b2e0ccbc605a56e4e776592df49
b21090e7b714b2519f970443edceed965f3acb43
48502 F20101123_AABTLK vanalstyne_r_Page_37.pro
c4858b51344bf7599e1f96bcacc357c5
7cbdeb354947b9b3b79aa0c7456b5b72eb42ee39
105666 F20101123_AABTGN vanalstyne_r_Page_55.jp2
321922cf5ea08ea13f0de7da865790d8
93886a9f46d643e381f788b943be1821cf6639be
18514 F20101123_AABTQH vanalstyne_r_Page_09.QC.jpg
866ef4c517ae281a67892a600a826b6b
6a3cbb0f217a649b3e1d1c66944cc7088eca4651
49619 F20101123_AABTLL vanalstyne_r_Page_38.pro
5517d356603930c6006292c01ceafec1
e9ec17e7f72ab30d7f8eb13f364d2cdf444ad96a
95478 F20101123_AABTGO vanalstyne_r_Page_56.jp2
f684af0c727fd8b3caca159c53420e2c
d08283338e055a20be68918edc60214256a9fce8
5254 F20101123_AABTQI vanalstyne_r_Page_09thm.jpg
ddd6be9605e594499a0d7d2aabc7177b
d40452b5c6536b94bc42fcd0dcb06c9a9e84fe87
24877 F20101123_AABTBR vanalstyne_r_Page_01.jpg
5650799c512a1156080dc3aaa00eb989
b2fb320717dc742f529fe8db55b6b896583976ec
51885 F20101123_AABTLM vanalstyne_r_Page_39.pro
393ea7bb1d29acf4056409972d5b5a05
0024e700d147202de752a8091a1b978bee5949a8
97031 F20101123_AABTGP vanalstyne_r_Page_57.jp2
b5507ab3ed06e77615e07654f113b522
5084b0d3990cfcf74ff970abd0c7eb3d60a91d5a
5803 F20101123_AABTQJ vanalstyne_r_Page_10thm.jpg
56883b6e25dc20a3be2f3d9ea0bf7b07
39a386adc5069cfe2c86d2ec2f78154b3ff147fc
10440 F20101123_AABTBS vanalstyne_r_Page_02.jpg
c0a9717ca9aeedb0af16a746dc117ffb
838d7986eee64dd70935e3438c1752290950c4dc
20504 F20101123_AABTQK vanalstyne_r_Page_11.QC.jpg
e94e6ec4fcc570fa4c5b0880be7a15fe
21016a2428caffd074efa0ccb4a20bb812f6ccaf
50325 F20101123_AABTLN vanalstyne_r_Page_40.pro
62342237e0aff36b72c68499a8b12bf2
5c4578810f69b351d277db72b52b896e31ea3d3b
109515 F20101123_AABTGQ vanalstyne_r_Page_59.jp2
4fe0e960a9650baaecdc1fbd051044bb
47794873d8052290d279c23635997a699584779b
11815 F20101123_AABTBT vanalstyne_r_Page_03.jpg
55523ba674c64434c4f8b0735e93cd9c
e73dfe584d5556358f3018b242fde0bfab6f5145
6035 F20101123_AABTQL vanalstyne_r_Page_11thm.jpg
0e112fe18fb76ce72ec75c8b0f92a884
6a6926293c868da2908a3b040807d7ae53263580
51448 F20101123_AABTLO vanalstyne_r_Page_41.pro
285217cfe96ca7836a4df8d4a8329ab8
82202b9940c6a84ffb1f790efce7ac6cc053c577
36989 F20101123_AABTGR vanalstyne_r_Page_60.jp2
1de18d4569dcf7de84d39edbe66f2cdf
0bd893e75ab1fe6c4c827e40579ef438b8c5a32e
22862 F20101123_AABTQM vanalstyne_r_Page_12.QC.jpg
8552c1206ac47468bd150a4716fa78f9
edccc0b1bbb33860ef012872289e0e146b2cc261
122371 F20101123_AABTGS vanalstyne_r_Page_61.jp2
bac42321b503472d634ef971392ff0c3
e230b23aa37bae2aa4dd3b5c76741e7e1db76329
62689 F20101123_AABTBU vanalstyne_r_Page_04.jpg
859ff1b982f3b2305ca87fa0ceac37cb
5325ce0e021650772d7b000d6ca956e510c69262
50244 F20101123_AABTLP vanalstyne_r_Page_42.pro
a9f7dba666b26fa6618087e41988ae56
08b8df20c94fc90b14ca8c568fa208faf3f1bce8
6458 F20101123_AABTQN vanalstyne_r_Page_12thm.jpg
c2c1dc8236896d3291204a04e75c9611
cbe4adb2628f32a48a0c7c5032ec459b33d27558
117088 F20101123_AABTGT vanalstyne_r_Page_62.jp2
83c85a072f78297d250719e38a915b59
043c72bdabfe4eb5d78892b6aaf7ecb4ceeb410f
24919 F20101123_AABTBV vanalstyne_r_Page_05.jpg
32464dc1a43146e97900936f94c06f58
f4587d9baf9083695749d7ed0f6393ba2449d6c3
50677 F20101123_AABTLQ vanalstyne_r_Page_43.pro
4fcf043d9e632b819e6ac5802c68df44
21aab4f955d85ac21e7476a8cc6a3f35679185ba
21746 F20101123_AABTQO vanalstyne_r_Page_13.QC.jpg
3ded3907b977883058feb8af11660a0c
b66f490d09841c81cdefd01fea13a3608fc11f91
95252 F20101123_AABTGU vanalstyne_r_Page_63.jp2
d982f3980063291ac23781cbcf7bbc6b
c1d656418531b632ebfb47dae0a897a549b32652
61407 F20101123_AABTBW vanalstyne_r_Page_06.jpg
68b139280867b74c455052139feae9a2
aef34d85df833dd3469924ea2fab3ecd4d427b67
50251 F20101123_AABTLR vanalstyne_r_Page_44.pro
8836715c2203fbd1cc95a3d9d060ebd7
59f8c93adbe49b091a95a3c0e8a52300482d9ed3
6225 F20101123_AABTQP vanalstyne_r_Page_13thm.jpg
e02afcc1d5c6d24931265cd330e1c2e4
13a441466513ad19d6f207025a282dab18db62ff
106000 F20101123_AABTGV vanalstyne_r_Page_64.jp2
6f44b202d9510b44f09c1483d764b3c0
7c30aaa5b074e089f2c862e144911c912d8fee70
48327 F20101123_AABTBX vanalstyne_r_Page_07.jpg
e99438c51293382c25ce3f9e23360629
09056ed4003c5dbb52335ed5471eaba6e292d9ed
50466 F20101123_AABTLS vanalstyne_r_Page_45.pro
483865493024168e1b1869a0b71c9e8a
b60c0e303e7d71da898d650a5d971748e878197e
20068 F20101123_AABTQQ vanalstyne_r_Page_14.QC.jpg
da2a3ded008ad158ee62a9a2dac9bb67
dcec8bb75fbc1e47501683e9bebde5dd5d6ad321
99909 F20101123_AABTGW vanalstyne_r_Page_65.jp2
cd0b936785e63438fed7024ac96cf611
4fd350e7acf555c4ab7d2fc89b809ffa93c2fcec
48829 F20101123_AABTBY vanalstyne_r_Page_08.jpg
dd0298e590d9abddbe2999c30b98f9a2
8a3d21aefb9adac91c4880c1550bf0a5026c3196
48565 F20101123_AABTLT vanalstyne_r_Page_46.pro
203fc255d63b6819c744df58b97d01d0
e086bf9088da7c42627685926ca65c309d5e6deb
5563 F20101123_AABTQR vanalstyne_r_Page_14thm.jpg
f1682434a612d26b1c143be101775a0b
d8e1205dd1cb83106ed54bef52bf7b0378fb7bf3
80419 F20101123_AABTGX vanalstyne_r_Page_66.jp2
928f0db62d1e64ad70ee7ec95c6b1e73
01ef985bf0e31487aca0dfc204bc6ac25e052530
59387 F20101123_AABTBZ vanalstyne_r_Page_09.jpg
42912f80e6aa07125ad8226bf128673c
60cda2ea92240b732546303ec1e542d469bf268d
23725 F20101123_AABTLU vanalstyne_r_Page_47.pro
93b5743741a15995474eded456d56e2b
b17621da0c5dfb5799c7643b496fbefc188fe31e
24098 F20101123_AABTQS vanalstyne_r_Page_15.QC.jpg
b87e4bb35c8822829bbae372764ab733
7629f35ee574de3fe63508b478d3bd041d15d19e
56985 F20101123_AABTGY vanalstyne_r_Page_67.jp2
00971a5d6c833ec5b64e142bfc1eab4f
d8acdc5ccb4cf6ae6d4217aebcbe048fe3c7f8bf
66291 F20101123_AABTLV vanalstyne_r_Page_48.pro
be90d4250be6aa7be877554b5a2610db
66b1a22e23ebab129736f3f0cae033b3f8ab3683
23165 F20101123_AABTQT vanalstyne_r_Page_16.QC.jpg
293f21250a0be68cfdbb6fb0ef0baf16
f358ea3e13931e9733f7ff46a4e2bb61c029294a
64148 F20101123_AABTEA vanalstyne_r_Page_63.jpg
399c31dbff3bbf06aef376fc1c74c087
6749b23ba85fd8567749e3ea3a93f1d874c0941b
58192 F20101123_AABTLW vanalstyne_r_Page_49.pro
b3c9b093db0a2ab81c848a8f027487ed
a3c7c3ae97b9793e08ae57d79161e7511718a5ea
6479 F20101123_AABTQU vanalstyne_r_Page_16thm.jpg
493445f6225328d6f6251c51408c19c8
038565c0c3d910a840ae313a61f51dd0351166d6
70920 F20101123_AABTEB vanalstyne_r_Page_64.jpg
dde3e85e243078c99c8a2aaafc48b49b
ff692838a1954227e91dd498f0f38ee376e79559
94136 F20101123_AABTGZ vanalstyne_r_Page_68.jp2
5345313eecffdd4f9231cfe987efb12b
b8d19dfcb5e6e732748508475937369291221c35
29756 F20101123_AABTLX vanalstyne_r_Page_50.pro
136bf309b0c4e45ef05d14c62820b0a8
031e7e58a3e1c2db265c3ec752b24f482239cfc9
23968 F20101123_AABTQV vanalstyne_r_Page_17.QC.jpg
c6130860d752df86c0169f4aeb3aa16e
4739d11fbe9bd7ee69057f0ccf00cbbaf8799db8
58787 F20101123_AABTLY vanalstyne_r_Page_51.pro
db7e2a4554883c81d45e70ad74529316
fc9737e24cae7704313032ee17eedce839590946
67476 F20101123_AABTEC vanalstyne_r_Page_65.jpg
984ea6558e0dedc121aa30ac5912a2f6
b48ddc9e0e833a389033ac12cc0162b3a00936cc
6648 F20101123_AABTQW vanalstyne_r_Page_17thm.jpg
b34524aba83be9a7850df5fab75effa2
44c9855b6d64fdfdc3a54dbdfb51908a75818e06
F20101123_AABTJA vanalstyne_r_Page_49.tif
1ebe9dce520e9c910aa81d8838ba0877
da19a13542f564caba55fcaa01534c4df2961fc4
19552 F20101123_AABTLZ vanalstyne_r_Page_52.pro
fe333c6c4d72fa5c3cd8bcb28e7ea27c
eb7a562006f420db90db5ccb60a4005cc4e74c23
54597 F20101123_AABTED vanalstyne_r_Page_66.jpg
09a45a9fa8ad3ea5643c3286d3ade8c4
74a70d88e3e6682cf55159184b934149078c46d0
22582 F20101123_AABTQX vanalstyne_r_Page_18.QC.jpg
1b432b8145d49aa27f8b2898529c231a
4de49690cfa3b9a51e4c2990515a8c19ec7e915a
F20101123_AABTJB vanalstyne_r_Page_50.tif
cb828ec00236ee22ff472a4885787191
1a780c11e840f9f25c6a33f8f46ed46b81d00d09
41329 F20101123_AABTEE vanalstyne_r_Page_67.jpg
b63d061d5adaa557effc34ff7281f527
3b45ac7210aaa9a3c53802313c20545f92db63a0
6473 F20101123_AABTQY vanalstyne_r_Page_18thm.jpg
83cf3c6d4ab954c98b73a84ec9952a06
a7d9f7911b23f7aaea49f37b1dfb36620871fcbf
1618 F20101123_AABTOA vanalstyne_r_Page_33.txt
1fc6d3d33201277749c1fe2911836278
f302da5a1db67cb9ef78e6cbc09420b7dab24d5e
F20101123_AABTJC vanalstyne_r_Page_51.tif
ef745f3a5da02f912a6b7e8e2ee0b522
74295331cc2e00e5bc1f7110c6562b2105571021
63755 F20101123_AABTEF vanalstyne_r_Page_68.jpg
a6cc11044551dd1432fd13d00be06483
3fe1b9b96f67ad0cebd136f1fd4760a11488704f
22987 F20101123_AABTQZ vanalstyne_r_Page_19.QC.jpg
5f92321760a47e36b42654e58e647d33
2d869fc411364e4b2d774f39524853572f552bd5
8423998 F20101123_AABTJD vanalstyne_r_Page_52.tif
508c2c075f5092dd8ca01a8e1289a5c9
f984c99b898d9e570920531c2b028b7f9f4ea3c5
78434 F20101123_AABTEG vanalstyne_r_Page_69.jpg
a3c47df5735e3813bb7e8425180d6db8
c8fa66bbf43dc484034fdca01a33e202f1e017f5
1934 F20101123_AABTOB vanalstyne_r_Page_34.txt
d344e1dff27b902bb93a7f5f0f1453b5
02537255e7fb718896b123e66e12af83bd64be35
F20101123_AABTJE vanalstyne_r_Page_53.tif
ddc8204b91f2d4cf8dba75138c856c44
118d4327e2758ff669a3fe705586cc2267efe171
81970 F20101123_AABTEH vanalstyne_r_Page_70.jpg
02038f89464f75afa880e5c724a849a5
b2c0e84b3f0c726957602ef448187da54cbaa05b
12612 F20101123_AABTTA vanalstyne_r_Page_47.QC.jpg
9706cc6fe3b2e3b17e0e5b6b027f4599
ab1f6b36263e280886bae38144fdefbade082183
1908 F20101123_AABTOC vanalstyne_r_Page_35.txt
4edebf8e6f4e6e01085d8d2ab9af1dc2
056c02a0be9c383fe32a5e7bf39459b41a91e588
F20101123_AABTJF vanalstyne_r_Page_54.tif
ac8e3644ac7892b3df0d385e041db47e
238d2a05ede04e5e2e66263a5c2f63f0d3d3fa5c
88151 F20101123_AABTEI vanalstyne_r_Page_71.jpg
205161c79f6133af3584f9fb93e73c5e
f8482b6e1b841a45881566d2adefa8a91212ae67
3687 F20101123_AABTTB vanalstyne_r_Page_47thm.jpg
49757aebbc742fbb6cd20360029d7478
fd004ede0280bcd302407d6b3f34230020eeac3f
1909 F20101123_AABTOD vanalstyne_r_Page_36.txt
c6a5e384aa69a653e08015aadfae429f
2123843eacdda006fe0bc68a0f25c78ef8a17441
F20101123_AABTJG vanalstyne_r_Page_55.tif
8d91b2573ca82834127347b069d97b83
43ea708f285c17934d5930e18eab55e6d2e24f34
48988 F20101123_AABTEJ vanalstyne_r_Page_72.jpg
c4941697803fa75d4f8b26c23380d7bc
7e3bfb7821c54ea0693000673d4f25beffe108ea
23254 F20101123_AABTTC vanalstyne_r_Page_48.QC.jpg
8aea9d03d2b22103e44b31d921c08390
9f9d47f2ee7e2898edf8ad6f3d62d24815c0f1db
1943 F20101123_AABTOE vanalstyne_r_Page_37.txt
3945784f92ac508175a5ba21ae182e02
d95f06d137e65c1d24083e4693b2fa43d45814cb
F20101123_AABTJH vanalstyne_r_Page_56.tif
2337205f6bd48fa8c3f707773fa75fe9
79a3df127463b70d00c96dfa4fddb7e484aa2c56
63766 F20101123_AABTEK vanalstyne_r_Page_73.jpg
c78d701b4680f9826c6850779f20afd8
55a9609d719565f27d59263c7fd1e1b356c39e90
5945 F20101123_AABTTD vanalstyne_r_Page_48thm.jpg
19ed29ccf6b0668227c084b572c9cd5a
fdb762faab30a1c84d4998d5fd5980229d5a0c14
F20101123_AABTOF vanalstyne_r_Page_38.txt
2f0fccd1fb6ae79d98e26c8e5a11c79a
55fa803abdb46de1a41fd4e0553a55af1dd34553
F20101123_AABTJI vanalstyne_r_Page_57.tif
6039a2010fbbbc9634a9358f11741312
702bf31fddd92741fd8eb4e9e404f77787ec71c2
28343 F20101123_AABTEL vanalstyne_r_Page_74.jpg
3162b4178851eaf8028455484b9f8138
9f95f06fde272d82d036775bac2c0ce6df50a411
22738 F20101123_AABTTE vanalstyne_r_Page_49.QC.jpg
355005230b8b27ba71c4af8ad8d50373
5e702fb510a42d3fe5d3562fc6a484472e86ecf0
2070 F20101123_AABTOG vanalstyne_r_Page_39.txt
4c13c4b988132ddfbc84dbdebb4acf3e
fa25225a79ddbbbdd88e0f6ea60f0988053a44e2
F20101123_AABTJJ vanalstyne_r_Page_58.tif
2c1262cd4e0f468d1d9592f111867f1b
c5c1fce40c70f07dd186ebb193871a7974366427
26414 F20101123_AABTEM vanalstyne_r_Page_01.jp2
19c58d0d220f2dca80e52d014fe70941
27cd60a518127c3b10e8c1224e05c04793b997b8
6163 F20101123_AABTTF vanalstyne_r_Page_49thm.jpg
3592b86d1adb7e8d76ed4eea6e84b218
47509521a6022a9a038e1e1af3fd5c3b408416d3
1990 F20101123_AABTOH vanalstyne_r_Page_40.txt
6e629d8b69281c7b0f6fa469bcdcb834
0d148a35768e385fa6ea3d0bfe6925bf4322dcbe
F20101123_AABTJK vanalstyne_r_Page_59.tif
f4ed15997fbe1c51e9c874bc56247f2b
fc10da494c1366dcf3f667d2eb5d1935141f49ab
5919 F20101123_AABTEN vanalstyne_r_Page_02.jp2
1560ea2ff46f157ebfb86b8ccaf1c643
f70057791a36a5baa407762a829b36cbe79a720a
2018 F20101123_AABTOI vanalstyne_r_Page_41.txt
c864a99d3890f4f70a13f2e41f354450
aeb413c9d8ee5c040bf8cf820f72be973736a3d4
F20101123_AABTJL vanalstyne_r_Page_60.tif
07d2977d79f90573b88058243621529c
2d0fb5105897f235ea54e81dc601d381457744d1
8429 F20101123_AABTEO vanalstyne_r_Page_03.jp2
bf77beac650f19a6c9e8e086b005792d
fcafa3e71c43f8856810616ea88041403cc19754
13047 F20101123_AABTTG vanalstyne_r_Page_50.QC.jpg
ca3dbe892ed39c69a4b6802393b9ff2b
7506668069d2e3b46f9a5ba31fd26106ada65816
1984 F20101123_AABTOJ vanalstyne_r_Page_42.txt
7df3b0d62a04f5961070894cfa8185b3
ab55daa366200fed00d1b691f150e62be86feef4
F20101123_AABTJM vanalstyne_r_Page_61.tif
85b4e5f52808259c52b00c2b5349ef4b
1264ece48b4dcdbc99a8aca880e521154297291d
92591 F20101123_AABTEP vanalstyne_r_Page_04.jp2
be97eee37cdc54488db4ff1e844fc386
acab761d5a2636800b043b96298ff89de589b8fe
21103 F20101123_AABTTH vanalstyne_r_Page_51.QC.jpg
836bcb8dee611786a00f68880acb2d04
df21a547cefcfee633f73b9db5a9260a20624675
1988 F20101123_AABTOK vanalstyne_r_Page_43.txt
7f5484ac2b48d9bb9bce0b702795f06d
628a9af2aee5ee11014a6ff1d7f28da97eea902f
F20101123_AABTJN vanalstyne_r_Page_62.tif
a3751a2230c2136e88298d4f903232f1
9f83cf4441381bf8c75944488b932418259f7c47
31469 F20101123_AABTEQ vanalstyne_r_Page_05.jp2
68b9ab4335773db03c2cfa9e624675d2
b851398f3cc676e6ed98affd7a1170346077e94f
6188 F20101123_AABTTI vanalstyne_r_Page_51thm.jpg
0c996966d53e777e8c46b6e14afae2ad
9d9426b186516c7426c9a94dc4f184407d29b153
F20101123_AABTJO vanalstyne_r_Page_63.tif
e74eaf5dcfb50e74839456ddd403a939
df4fe14151bf14aa6fad1b7607f2646f9f68c152
1051961 F20101123_AABTER vanalstyne_r_Page_06.jp2
ac15c1ccc97ec910062dfb1d136ca65b
c5751a5066a7c310d7ed9eef7c879bbe54d2e9a7
1973 F20101123_AABTOL vanalstyne_r_Page_44.txt
9e2c6880e251d919714f27fc48832aab
e7fe8a74df1e000285c0ac7d9888a68282af26c6
10786 F20101123_AABTTJ vanalstyne_r_Page_52.QC.jpg
ba9db817848e16c152e26b9f42842a90
a0b543fe01af6c098df8b4b2f870c98db16a8db6
F20101123_AABTJP vanalstyne_r_Page_64.tif
7687c2dd926a11f321e983af155f939a
8697bb0025f991c6607c0b76724ed0c72f8f695c
1051979 F20101123_AABTES vanalstyne_r_Page_07.jp2
f958550d7e5e7951330254d3abf59c23
d92d20f1bc4ba1d54413b484d71b3ef4dde30867
1981 F20101123_AABTOM vanalstyne_r_Page_45.txt
f0ff85424531c2f521261fd5ac916060
012a6b85548152f080da22eda1e0f2eae313cc9b
3150 F20101123_AABTTK vanalstyne_r_Page_52thm.jpg
145ccf39943888a2394a24fd1574631e
fecdbde941bb1781a2f5abd430d7f70975717b6b
F20101123_AABTJQ vanalstyne_r_Page_65.tif
526ddba873856b5cc2fa004d70689e01
13e86b73bdf992f9442b888dc3c828dd5e0f8bc3
1051975 F20101123_AABTET vanalstyne_r_Page_08.jp2
6d9431260ac3ded80507b0574821055c
048457daeef3ce9fd298f06a80bd0f2228d89d6f
1944 F20101123_AABTON vanalstyne_r_Page_46.txt
dac1574328c5229f02432d612f73ebc3
32db89f53e6f8aa48d1fa94b5f8075304b88fddf
20648 F20101123_AABTTL vanalstyne_r_Page_53.QC.jpg
4a5c274ef3ff4653e7ec70d3e2d54fd9
9767f49c8809c324c2d533a36cca8eefa5c0045a
F20101123_AABTJR vanalstyne_r_Page_66.tif
104b7afa77ca0abe6dfd2b36c5b3e950
d65400d53e9936e716b3f9862ac4b4ce0ff4cfd0
86447 F20101123_AABTEU vanalstyne_r_Page_09.jp2
c2f3ff52ce5b3bdb852507bc9c591eee
a560a6357858f157d164277c5030a475bcb593d2
985 F20101123_AABTOO vanalstyne_r_Page_47.txt
aea87cdc9c5ff825c84b0bcd897e500a
2a140d201185b24fe577d2ad22b4c419cddf16aa
24368 F20101123_AABTTM vanalstyne_r_Page_54.QC.jpg
92fd940097c626ebf46b6308c6cfb6ae
1cdc0ff366eb443c616a412d754bcba0c6058178
F20101123_AABTJS vanalstyne_r_Page_67.tif
07368f82a49c669df65b02290fc18f59
3411c85a68f5066ca8615262359a7b6c9d7d28a9
94768 F20101123_AABTEV vanalstyne_r_Page_10.jp2
b044a20a2f01a2367f06a79f56749c4b
910a389419ceccdc9e960b60571c63a8402d9341
2909 F20101123_AABTOP vanalstyne_r_Page_48.txt
3d966b0db2b582890f787706d654ebb8
a7564dfa6bda04a9a2644309473e8646b28ec08a
6767 F20101123_AABTTN vanalstyne_r_Page_54thm.jpg
149fb4d5b6ff72467d494fb03eca09c2
135d95366dcfc4cdc510f93d286d7485bfcb03b7
F20101123_AABTJT vanalstyne_r_Page_68.tif
06f97d7adf3e3e4641b09040bd6e2be8
b814efb9e9d451eeb89cbfa10261c04fb378337c
95020 F20101123_AABTEW vanalstyne_r_Page_11.jp2
f46f77fdfcd01718a4ffaa6a740db6f5
35f3b009cdb1bcca05b140bf87a715b0d2f26ebd
2664 F20101123_AABTOQ vanalstyne_r_Page_49.txt
778b5197358f712d25144ab3ffa10e6a
e24ac919fd87a3eebc3549266d5fcab6e30d65c7
22784 F20101123_AABTTO vanalstyne_r_Page_55.QC.jpg
d7e81004ac5a126f95565b9ce9d8405a
58db17f69d97f7364e5b3092713aa0924199d098
F20101123_AABTJU vanalstyne_r_Page_69.tif
28f09da84c6d69e188720091d2b82ecf
cf7ee9df160447b51a5bd4e4006dbaea7ec9cb5c
1423 F20101123_AABTOR vanalstyne_r_Page_50.txt
266558ec6fe0a42dcc62b82b05d190b4
9a27d68f9efc1a7661160ba431274fb1a46bd8a1
F20101123_AABTTP vanalstyne_r_Page_55thm.jpg
4683bb841393b6331a6342579ed892af
180e54361269c3842278a671b4b19f8c107dc7cf
F20101123_AABTJV vanalstyne_r_Page_70.tif
753e3b5022f7abdc9dd0dd8a6ef5d830
afae167ba3ebe3a79abaa8892aaad4d3d2424c92
106576 F20101123_AABTEX vanalstyne_r_Page_12.jp2
31f8ea2957dcc07245b8e960ca489eee
f7f0df2030eaa3438c31cde5867574039d8cad93
2622 F20101123_AABTOS vanalstyne_r_Page_51.txt
e5a7c261a636968cfde568e5feea24d8
475b871cc05a8457965190ae054597a85e0d1163
20499 F20101123_AABTTQ vanalstyne_r_Page_56.QC.jpg
361f63e9b1bc3d5552fd9c64b676d621
2769f58771a53ebd8c362053b45b8b6aa1886598
F20101123_AABTJW vanalstyne_r_Page_71.tif
7c72589c8a02f885f0750f9df902071d
66c81eb6c7178c293ccd4dbcf31a7cd4fa168745
64283 F20101123_AABTCA vanalstyne_r_Page_10.jpg
76c7b61a65fbba59d8c5dae47d319349
0222d2fe9cfb14b41382c1d8d93b85e26060c752
99140 F20101123_AABTEY vanalstyne_r_Page_13.jp2
d1a83f734d95bfb9574be72c406aeac4
fcd5cb025b91d4433047aa9f40def41ef9e0e0eb
892 F20101123_AABTOT vanalstyne_r_Page_52.txt
b478382452ca62235f40948fe61f37f8
10218ddca2d55e30cf140a57b26640790fb6fc90
5942 F20101123_AABTTR vanalstyne_r_Page_56thm.jpg
51a1d7f9ef4fb13f6723f4cc9c1b99f1
51f847995bf65b997aee512622054fb96a97bb0a
F20101123_AABTJX vanalstyne_r_Page_72.tif
a44aab15342cce693b2b12f76a08835d
04e5404f0ebde10170d55ee468d153400fb0bc15
63471 F20101123_AABTCB vanalstyne_r_Page_11.jpg
17aacca5db8ed17a08cd073f006ec55b
643fe8e78c72a6f2cd32fbc878367eb0a65b0c1e
90191 F20101123_AABTEZ vanalstyne_r_Page_14.jp2
b4c033c4d3e2cd1ce8eea2df80e0a6de
05183ea25861165395f528b6be430ae0c2bee4ca
1749 F20101123_AABTOU vanalstyne_r_Page_53.txt
978eed2dfe4a5fc32bcaec79ae914aea
d460cb8a02a2da6673a02294b5f66ee7a249627c
20610 F20101123_AABTTS vanalstyne_r_Page_57.QC.jpg
e10de0e32f829af93e132267683b7cca
51694ec77748590876e5de4e0cc0b26ff3cc449a
F20101123_AABTJY vanalstyne_r_Page_73.tif
a17d8249feebde1cd6d121628e2040e2
b13db12dbba781e3079033c351d1df172afdbe90
70835 F20101123_AABTCC vanalstyne_r_Page_12.jpg
1a6e6a38197a7e3df7ffad761cf86d9c
1cf09b866dad15a4409220e68a435244c821958b
F20101123_AABTOV vanalstyne_r_Page_54.txt
1ace465455831c0e5632ffc26b59fa36
9c52fba51ced3533cbcaef18213898a73cbfb4f0
24423 F20101123_AABTTT vanalstyne_r_Page_58.QC.jpg
36f2e9184c843a39d54895bfa60e1bec
1f7db735b162efb4856808dd6f0cfe302e0956d3
F20101123_AABTJZ vanalstyne_r_Page_74.tif
6238a4dd4a19ef8044ac85871fa94f58
e325771909e08ae04babc1bd429b4ee2b720d1bd
66267 F20101123_AABTCD vanalstyne_r_Page_13.jpg
3f3ddc6bd0586ce51335f9ed7d67ed08
082d0f40c0d7e14e0fdf581520f4d3a9a81f31d0
1907 F20101123_AABTOW vanalstyne_r_Page_55.txt
843cf452ec51cb329b83ea65d6ad1445
68013ecc49026b89bea44771385b902f2e62eb79
116774 F20101123_AABTHA vanalstyne_r_Page_69.jp2
3f2cc825544d97c42e0def2adf78d442
585d0d5aa98335225157c8d43866a948e08770ec
6830 F20101123_AABTTU vanalstyne_r_Page_58thm.jpg
f76900c02f325f89646cf509298d6b6b
ea138c6320f9228529e40a03428aeaba0f059f80
61211 F20101123_AABTCE vanalstyne_r_Page_14.jpg
3c155f04084c277bee65ad317a0b4f92
7037a26110a200e8051d267e98e9aa095b237a5c
1698 F20101123_AABTOX vanalstyne_r_Page_56.txt
c972429a743d0b14c7091aab7f8928f5
64288dd60df8e396aa55e6d51e6096f25644944b
122451 F20101123_AABTHB vanalstyne_r_Page_70.jp2
aa9aebaed4321afea2b2e867771ed17e
2977464e03167ffbd54c57fb94b45e553a30d74f
23836 F20101123_AABTTV vanalstyne_r_Page_59.QC.jpg
b16293e80763fc4b1d70213280d165a8
fa3d37f42812b3bca0442029c4f12e34f83555c4
75251 F20101123_AABTCF vanalstyne_r_Page_15.jpg
600f0987010e5a3ae7051ba1f58e441c
663e0b942b5c22c7abeda933472d2ba5c1436857
1739 F20101123_AABTOY vanalstyne_r_Page_57.txt
5ed5675cc95794d2cfafb1def4df40e6
3804e72826e8b85f4bf4b882256bb439f6d242d5
124699 F20101123_AABTHC vanalstyne_r_Page_71.jp2
1a96d6022c4a19dd55e6a6dcdbbb00f4
b10f1cb10275e176e65c847320b99432c6d18a14
6512 F20101123_AABTTW vanalstyne_r_Page_59thm.jpg
8011995dbab1d9af935a7d6c1f96f279
a3473ee43bd81b6f80377e1dbb7f41d7841a81e9
72004 F20101123_AABTCG vanalstyne_r_Page_16.jpg
a915b0edfd002255d995260c9aef505a
3d376d4261b56fc8d4278c84ff6b2414f2d41f7c
42270 F20101123_AABTMA vanalstyne_r_Page_53.pro
1b6a7e5b68b36e9d129a079a38bc4d2d
254a9040c1c13f007ecea96e5110dd79abdeb020
2072 F20101123_AABTOZ vanalstyne_r_Page_58.txt
fdb0c9e0413fddeefe4f292c037d8149
4b78afb3e553c627bf9157c5e412194b46f44a06
68743 F20101123_AABTHD vanalstyne_r_Page_72.jp2
a33e85539c1b30dd042941929e3a98f6
5c53db95051fc41ae53af91da72c8526c7653800
9219 F20101123_AABTTX vanalstyne_r_Page_60.QC.jpg
f4ee2ff92bbed5875ccfb521b2b97883
d404ac5f43acd429563fc332558441379f12de16
74029 F20101123_AABTCH vanalstyne_r_Page_17.jpg
a2b6b2570ad5b3ef497af8cdfc23d0ba
e76378a3e1b917af1ce29e6b1e8640ebb3e71d63
50578 F20101123_AABTMB vanalstyne_r_Page_54.pro
f619b96b4300b5cdfeb265b80efc4abb
ad8f089d0684eb46c1b849aad22640122a594116
94467 F20101123_AABTHE vanalstyne_r_Page_73.jp2
e4f8116f7b3f8857ef877d1bacfdd02b
eb56d18c336702dbeda0a609c393bc38ae4f5b93
3066 F20101123_AABTTY vanalstyne_r_Page_60thm.jpg
94381f6c12f53fefc62d563c16df8679
51d2431f339896b3ffd736547e9019aff9ef4899
6356 F20101123_AABTRA vanalstyne_r_Page_19thm.jpg
f4518b3bc951644e83e5f835299c5570
39f039047060a355ad2c570bbc025b2177879684
70513 F20101123_AABTCI vanalstyne_r_Page_18.jpg
2de54bca0980a75c5ac7b2b3ebb40e76
b496bf35a8d2683b2e8a1c75bdb89a06c5f34209
42620 F20101123_AABTMC vanalstyne_r_Page_56.pro
75dd9dae9f350abe01d90a5604565f2b
d22b290224566cf66d6d59406feda1b9b9f2d581
38524 F20101123_AABTHF vanalstyne_r_Page_74.jp2
a985dfb21e3cc184e1aba73675d95d2c
4a3ec086ca82d0309187f61188fffb48a4d5efb3
23332 F20101123_AABTTZ vanalstyne_r_Page_61.QC.jpg
cc09517c6b852550e0edf0a0ae1dd79f
43f7bcb1ba53418b0964d7896cef6273ccdf1818
23130 F20101123_AABTRB vanalstyne_r_Page_20.QC.jpg
38372b4baf62f893281dc14bd18ee1ab
8f35d54c2a25da05615478b83b487749dbd91627
69498 F20101123_AABTCJ vanalstyne_r_Page_19.jpg
8adeb37161fd0bca96ff3274a034165b
7df10a7813ac77c80e1277fc0d076a112719614d
42706 F20101123_AABTMD vanalstyne_r_Page_57.pro
b57cccb827bdf67a2b20db8d829c1d08
04225556229912675817e284334eceda9fd892b7
F20101123_AABTHG vanalstyne_r_Page_01.tif
76ad04237442d69f692a3f797a62f44c
b142bcc2d990d85047e366c2ac9784fe91b9e854
F20101123_AABTRC vanalstyne_r_Page_20thm.jpg
7d888583661a8cbb5886a7142918483f
38b8d19d4aa213400e2549d3315ec798c2cf8414
70774 F20101123_AABTCK vanalstyne_r_Page_20.jpg
fec637457441ee4b05ff1959f68a1f49
34d20f28d365d6efc98fcd93880769007b79ae40
52051 F20101123_AABTME vanalstyne_r_Page_58.pro
01b18b306a00734d9d7af06e1b64d79d
8ae9c6028806caeb7d360781df58f02262011945
F20101123_AABTHH vanalstyne_r_Page_03.tif
54345208bd1ab241ebcc2a914131b577
41940a959d713849b3b7e15960c09bb21cc7429c
23196 F20101123_AABTRD vanalstyne_r_Page_21.QC.jpg
8a67799aab1a10353aa79f83b9e4302c
0624eb6e90624251d4a49de90d35c74f42c926f9
71568 F20101123_AABTCL vanalstyne_r_Page_21.jpg
593acddad3c1e52669a73a24987e5e04
b3127a14d516cac8dda16842fb5ed72d12470967
49182 F20101123_AABTMF vanalstyne_r_Page_59.pro
0102e3b973c4a15033234742ca68f43a
cac12f926092f5b1a91bfdf2469b20b16931507c
F20101123_AABTHI vanalstyne_r_Page_04.tif
d8bb5be479af492bb5dac8b52d780367
1b1d0c3fd96e0dc0dbe151ed8ca334e5addc6f8b
67258 F20101123_AABTCM vanalstyne_r_Page_22.jpg
ccd364747ff3cfa55c6c540e84e3e324
31e323b851cc60d6c72034a1dd38eea023033972
15099 F20101123_AABTMG vanalstyne_r_Page_60.pro
23cf50d171cdc16f4a099e8e0c15cfb2
6740bc5c78518448f73c659c523ea3b789291fa0
F20101123_AABTHJ vanalstyne_r_Page_05.tif
4fb332071b720674912627074e662717
3f20419f4ba02b323322a7715d6d9815bc7a23a0
6417 F20101123_AABTRE vanalstyne_r_Page_21thm.jpg
e5b63c8c4f95c4c901575e01c1569aef
bc0cd469f04d53c2eab0059b56598b84e189e664
69265 F20101123_AABTCN vanalstyne_r_Page_23.jpg
a765ca7433aa110f90f3cf21e4a61109
121a2d1187bc3498f5163c70ceca949439612974
66214 F20101123_AABTMH vanalstyne_r_Page_61.pro
aa02fb22514965754c269af00b8f01a0
01101568d8089d4566dfead24407fecd1db935d0
25271604 F20101123_AABTHK vanalstyne_r_Page_06.tif
85d3ce5517a487da02621d5324fb2d94
0edce92820675c5e64fa8ed754cbaa901b3860b3
21543 F20101123_AABTRF vanalstyne_r_Page_22.QC.jpg
237b29932293f9d9795874ff56ee5161
d72fc3dac1446f6851213a0bb98f96ba6b096ad9
73128 F20101123_AABTCO vanalstyne_r_Page_24.jpg
51bc485edab8405b8a4709f95de6bc58
0593d10cfa42c96752d137a82f30ac85cd60cf10
60309 F20101123_AABTMI vanalstyne_r_Page_62.pro
e860808bb5c2c9d0478da5e20246027c
c9481538cdb579e89220c4e0af2004d5e8d3d103
F20101123_AABTHL vanalstyne_r_Page_07.tif
e402d68af634a6396c8dc67acdcef901
820d31db13966a85f6fe253a2bd4f0cef39f4720
6419 F20101123_AABTRG vanalstyne_r_Page_22thm.jpg
7e629cc59ebe73401097bebc3f1e5f67
1fad1895ca3f5cc089d152f0e3b9110f7fd49454
70236 F20101123_AABTCP vanalstyne_r_Page_25.jpg
71855bf31b24702d6cb3f1fec4a0d2f1
93db1fd9868b0c387e486dc946b49b749d5e7c28
43675 F20101123_AABTMJ vanalstyne_r_Page_63.pro
a5c26c70afb7bf57a784b1ad3bc0b59f
e152d0215bd94ab1cadc6c52ef1acf5ff47c8dc1
F20101123_AABTHM vanalstyne_r_Page_08.tif
cf714f321c0529ccd49286c3767f1b2c
18a0ed87b3521465a9bb770f6945c9f350ad76fe
22435 F20101123_AABTRH vanalstyne_r_Page_23.QC.jpg
339731854242be685583042f170706ce
31dad2d34d1903fdc9c7ac5cdf9fb40ef9642a61
74423 F20101123_AABTCQ vanalstyne_r_Page_26.jpg
a85b8ea1c2ab9f8fa635dbf98e9d47ed
94d12dd92fadc05edc625d989d467ddeb2589c55
47594 F20101123_AABTMK vanalstyne_r_Page_64.pro
da873fe031e22f984172f9dc274d9663
66e0b5c037f14e2235ced9cece34e575992fee6d
F20101123_AABTHN vanalstyne_r_Page_09.tif
fc526913b8efa02d7589c0391a0d6778
d3712d061b77d8eb9b318862fef78adebb6a0bbe
F20101123_AABTRI vanalstyne_r_Page_23thm.jpg
6f36779630f6761e3df861adb87d3b95
a6edbebcc494b02cc11f25f472cf7a09e55c3599
70531 F20101123_AABTCR vanalstyne_r_Page_27.jpg
dd5ddee220363596fd64e0c1e66d8937
b20132781c704ac59cd057a4758bba80f79f1fa2
45135 F20101123_AABTML vanalstyne_r_Page_65.pro
5b1c8f789203cf91b95fc5e380359e1a
a65aefd73a2f41c3ccfde24170ba9608e52180a6
F20101123_AABTHO vanalstyne_r_Page_10.tif
5ba35d5d07135e8536522e480019edab
6967fdb78354a7f89f51ee09b81e41c5a4196e84
24203 F20101123_AABTRJ vanalstyne_r_Page_24.QC.jpg
9779a8af85951c80f76282233b928e7d
56c3a25c6f3bff2589540708b1a1ec41bdcbae32
73953 F20101123_AABTCS vanalstyne_r_Page_28.jpg
d768a4b1ed86cf4d88881eb5537142f5
d91fa49a1db879fc8ba277b61127a0ae013573b5
25679 F20101123_AABTMM vanalstyne_r_Page_67.pro
7839a53f07e921e0ced0c430249dd75b
53a90e7a9569a88a0fa46d37b87385519f2ad684
F20101123_AABTHP vanalstyne_r_Page_11.tif
76d3c4a74b3a2373c7655a8a35efa630
5fa78296f162e39b824755c853b2d037bb206f09
6744 F20101123_AABTRK vanalstyne_r_Page_24thm.jpg
210ec7f3577add5043da00088f811529
c240eb5d667b806fdd3d73df5d10f89c069eefc0
69321 F20101123_AABTCT vanalstyne_r_Page_29.jpg
2040fdd0873ff6d821d42c4ef1176140
c12fd9953cb56056e665683d498562f68c16f8b8
44665 F20101123_AABTMN vanalstyne_r_Page_68.pro
104dafd7199833f32e2cd332a2c9d469
7616c786cdd588254cd91e9f290657e95720c998
F20101123_AABTHQ vanalstyne_r_Page_12.tif
07cfec44b7bc5e1bc434accf9c3432d2
c84667cda4bfbc4f4bda116c99300bf17e97cb29
22644 F20101123_AABTRL vanalstyne_r_Page_25.QC.jpg
5f0a7c812514fc932a1b8546292b2652
e9b9398e919ffa07d7a829d465895082226fbe65
72018 F20101123_AABTCU vanalstyne_r_Page_30.jpg
a502fa7e84147ce99df6254381902eed
97c9172ab33e6ad3251a0b96514586ed8db72627
56044 F20101123_AABTMO vanalstyne_r_Page_69.pro
e740794620e9d12ee37a5d62ab057ff9
b369013177c3e05ac12126807e593bab007aa107
F20101123_AABTHR vanalstyne_r_Page_13.tif
be4ffb68f07a5ee69409e6a0f1eb5bb3
6277cc1c01ed837131782eb4610585f73a77ad37
6389 F20101123_AABTRM vanalstyne_r_Page_25thm.jpg
59d36bcede15fc929c134efaad7f3120
9ef300fe6713c0c5d7d3aab5913f25c5214378dc
59659 F20101123_AABTMP vanalstyne_r_Page_70.pro
5c23d7e77004b39b8a3691fda28e3808
278195e3c483c9bd29378d24f7be86b251a30895
F20101123_AABTHS vanalstyne_r_Page_14.tif
5f79a619651afd8f8ee51e08a41f40bb
126f8f34e3163090dd1b72327934dd25e4eb5a71
24000 F20101123_AABTRN vanalstyne_r_Page_26.QC.jpg
a650fe1b7d0cb07d129da3050b8db8e8
fd27401f7b2407c619f96ac9cdc2481c13a4477d
73546 F20101123_AABTCV vanalstyne_r_Page_31.jpg
455991c9d3bd3fdc8d528696e7e64355
f16524300a4a32a92f8df101e3bbf571cf64d862
60303 F20101123_AABTMQ vanalstyne_r_Page_71.pro
014e8854a59ee7ed200e771f38bdef16
4b0856123ce4617a31da452c70f8567c4de61453
F20101123_AABTHT vanalstyne_r_Page_15.tif
4c1e5ee347b55770ba7cc26558e20806
8e2016af9e25904a9fd52948c21d9b0efefb0cfb
6720 F20101123_AABTRO vanalstyne_r_Page_26thm.jpg
416ef19b8ee7bdf0315cec9e8015f23c
677fd37dc26a6de57b5c1a4221ea07adb3c65e9f
25094 F20101123_AABTCW vanalstyne_r_Page_32.jpg
22da559ed08139f020ae51208b99b8c7
ecc03b0d045dc960439491137190552be980db93
32525 F20101123_AABTMR vanalstyne_r_Page_72.pro
e879def630a8e14ba81607b6758bde0d
e24b52001d8ff9c5cb4266f92b9f20e5a87ccc70
F20101123_AABTHU vanalstyne_r_Page_16.tif
27de80fbb75a75aa1224543944ca442f
97c6add194718da023da7873b1b492e2639e036d
22907 F20101123_AABTRP vanalstyne_r_Page_27.QC.jpg
afb173942c455e48441d8e819991dc53
fa4c4db3668aa3996951a794280506ba3985c2f7
59552 F20101123_AABTCX vanalstyne_r_Page_33.jpg
1ef26a7424074ab73af6886df58f6689
14984ef231e647932c18177361b063fc89e26d84
43587 F20101123_AABTMS vanalstyne_r_Page_73.pro
a7884456ac3bb770530693deec193c04
f3725e47c86eb0fdd5cc67062a3662499e6818e4
F20101123_AABTHV vanalstyne_r_Page_17.tif
a79411ac191c38c2a431150214c1eba1
9d3902561f1599c9a98c4581756ed6a73ab685f8
6337 F20101123_AABTRQ vanalstyne_r_Page_27thm.jpg
ad863f6bb71d9df76398fd82bfcf1d8e
613a711f241a3691fe34ca2d585b16bdbf983eb7
69398 F20101123_AABTCY vanalstyne_r_Page_34.jpg
78b773790863fba2e82682ccf5e245f2
40116a401b1f742f8b9cd1178d1414fe697fbe9f
15935 F20101123_AABTMT vanalstyne_r_Page_74.pro
9ff2eab73452d4701a54c017048b85d4
826fec19eff8da311ba955bd21cd52e52617c0c9
F20101123_AABTHW vanalstyne_r_Page_18.tif
538256a2e17c007c98f426dec25263b4
2d7d167dad6af6731df0b8b3f2e99f2a9ea301b3
23985 F20101123_AABTRR vanalstyne_r_Page_28.QC.jpg
cce4dbcb9465d41fc842d242c081dd8f
7789f836349d033c861c76fa3c1aea245ea529b3
70851 F20101123_AABTCZ vanalstyne_r_Page_35.jpg
afb825ca1f76f119744df0b2c7cfc988
294768f9306bc117f3ccd15c2d862cecda6f8a5c
476 F20101123_AABTMU vanalstyne_r_Page_01.txt
50d849f375be94c1e30a28078b9be968
6c573ccfc8acfa3c19e2aa24b6761a65568c40a3
F20101123_AABTHX vanalstyne_r_Page_19.tif
3ecf7b630536c0add911b5e33d927ab3
1db8292bc836177e3a530ae266072078b5e349c6
6710 F20101123_AABTRS vanalstyne_r_Page_28thm.jpg
3b19390d2d16a56eb5d1393e765e1b1e
b61008fa105ee08149afb2403999feeed5906e84
116 F20101123_AABTMV vanalstyne_r_Page_02.txt
a02b3992f49cbbbe38c11daa0f1d594c
2255b80774d5876cd241ea8e092a0f9cef828d65
F20101123_AABTHY vanalstyne_r_Page_20.tif
2838c8bbfbf706f5d6b673cbb997c18d
de5454b93e5c2724f6b81c4d8ecf2cfd8d3e35f6
22920 F20101123_AABTRT vanalstyne_r_Page_29.QC.jpg
7adb9cb11b30662f81061e8df15d2f8f
6ff2ab4897ef25cebba4d84e21ef1480508e3c6b
146 F20101123_AABTMW vanalstyne_r_Page_03.txt
f9bca261d0758833a4e58ea49d4a782c
489bb30db401c32b8dc62a5e9dfaec3b278dca87
112170 F20101123_AABTFA vanalstyne_r_Page_15.jp2
e200c91ec7d451ba1b700c374b176a14
03af755b42ae76e0a6c9109acd2ab29821eeaa22
F20101123_AABTHZ vanalstyne_r_Page_21.tif
a3565502b94343bc49f6174ef60192d4
6543d7cb3d46666fc4334416f6fdb254228f4302
6443 F20101123_AABTRU vanalstyne_r_Page_29thm.jpg
897a5f460d4ae3fef994fc7e5450c22f
6fbed8438a58709e91952da9757e8d208c9b3a06
1730 F20101123_AABTMX vanalstyne_r_Page_04.txt
50968ca3fa6cf7152bb15dcd7b0402f8
a48151d9d8f1cc84e0e08520aff660e7415d24fe
107862 F20101123_AABTFB vanalstyne_r_Page_16.jp2
a30786c7a7bab458ae41ad79719c1798
bd4f8d217e49a967bd573b9e9349f60beecd14f8
23626 F20101123_AABTRV vanalstyne_r_Page_30.QC.jpg
7ea7a7c4602375960fc6850bfd39bcbb
a695bab4e3ed77c3d232eb9d6bd5b7a229939fe9
537 F20101123_AABTMY vanalstyne_r_Page_05.txt
c3fc36e9cd76ace6011d032d64e5eb09
0068889753e72c6a134f0cbd7113d47432fa30ac
111212 F20101123_AABTFC vanalstyne_r_Page_17.jp2
2de3be417cfb0badd7325782dd4e8a77
8afd46105fdf5c1f95b8a86e2b70c8ed5e2182c8
6616 F20101123_AABTRW vanalstyne_r_Page_30thm.jpg
f9e8b5d106801cb1fc079d43d657af24
a3bf92b5187878b3e9195b544caef9d795f3e11e
2716 F20101123_AABTMZ vanalstyne_r_Page_06.txt
3792b598b0b6cc5d954317d033e0e77c
9c0102642c6bcde30adabd6ee1dcc836495bbc0f
106784 F20101123_AABTFD vanalstyne_r_Page_18.jp2
15e1c0bdfe58e23b50e6cf6bce0cd05b
6282c5ae340b853d9630372a08884b484896ca1c
9028 F20101123_AABTKA vanalstyne_r_Page_01.pro
275c13b0ba6def29ae145527adf95261
cf92379ebb4b9ba1bfa4f8e3423792d27e61c141
24076 F20101123_AABTRX vanalstyne_r_Page_31.QC.jpg
9a1d3a6fabb94fdeb9a66c7491fb784c
b11e72be9c1516ef5fd7c339938c95c08ef6657b
104513 F20101123_AABTFE vanalstyne_r_Page_19.jp2
c0cc8ebf4a12ae6b8b381846064fbb6a
24d1a4e9e515a0373517a4adabcf9045a73f1d71
1255 F20101123_AABTKB vanalstyne_r_Page_02.pro
f765f9b938930e20f14ae3206f68a273
a208ee6f51e7c444b001461a57188100ec50546a
6611 F20101123_AABTRY vanalstyne_r_Page_31thm.jpg
dcc102da95e5deb3f2cff4c5bf431b11
3fa5f062a32011a1199a350e08a41f99a1029a11
107843 F20101123_AABTFF vanalstyne_r_Page_20.jp2
e69987957777860240896bb157eeebb4
ebb4b8c51fdc6dff578cde0d461c0afcc4b558de
1963 F20101123_AABTPA vanalstyne_r_Page_59.txt
823b47ddee686e8aeade2cd18e174599
8851f842afa39c28eccb3a48059c85569ec6aab0
2343 F20101123_AABTKC vanalstyne_r_Page_03.pro
d9b3f5c775bce384ce1715889cbc20fb
7c4170275e4cf8c02736c11258aadd1388a90a96
2631 F20101123_AABTRZ vanalstyne_r_Page_32thm.jpg
3d7a3c08da05b43936e68fe94479f997
8f16f20c76978a34914b6c89fc15443134a269d0
106591 F20101123_AABTFG vanalstyne_r_Page_21.jp2
d83deafe8d8ad0bd0a54e4e277a29139
f11e2444dc60907d947e4de921c6b70b4dc6c979
646 F20101123_AABTPB vanalstyne_r_Page_60.txt
f3d0525375acc023ba2f424891f31d88
37ed89f9074f587fc3d1c756ad47f1ef6f706243
42842 F20101123_AABTKD vanalstyne_r_Page_04.pro
62eaf4e29b32dd138f307ec713a81be1
074caf0d0f9188cbca2d3270b5475778facfd8ef
101066 F20101123_AABTFH vanalstyne_r_Page_22.jp2
7ea6b79416cfe1ff531f3fef1a594c16
32e17537af05bb69c5680b4914e534e09b67689e
13494 F20101123_AABTKE vanalstyne_r_Page_05.pro
11af83e0e2af4b64da436df754beb232
d2b25b6fbb0667b1c9a9ebb6c41eaf4619c8e2cd
5952 F20101123_AABTUA vanalstyne_r_Page_61thm.jpg
132dcfdcf2340360e0312dcb0cb6f263
910cdcc4f86c9570933ba1b7238ea22f79ffe915
102624 F20101123_AABTFI vanalstyne_r_Page_23.jp2
0db5a849c72da896ca02d861aaf5cb08
ace11a163071f19f24b22e6c313a185ccece768b
2955 F20101123_AABTPC vanalstyne_r_Page_61.txt
adc5df8b9fad8bd9fd7d02d5b389cadd
52a0ed61ad73259d9650f987187a3446a88dca62
65499 F20101123_AABTKF vanalstyne_r_Page_06.pro
6faa281ff6409e1dbdcc3484859500d0
ff4728a6b61212609f8447cd415e90ea76d3702c
23138 F20101123_AABTUB vanalstyne_r_Page_62.QC.jpg
60de52fb0a005019dd8722bfb62ce0fd
24de337fee8055bf5f5eca62c0ed684453c2514a
111219 F20101123_AABTFJ vanalstyne_r_Page_24.jp2
cf5a36c175c45a9c269a709cc541b1b4
762ebf2ad227dd26f3d0cdb60cc0b7159ad47102
2940 F20101123_AABTPD vanalstyne_r_Page_62.txt
399677a27675e9b242a057ae7c96e668
f3344a0e77d064446c3b0220341a80795000c7ab
48755 F20101123_AABTKG vanalstyne_r_Page_07.pro
49b16e5be10f657c475c73fc0a6f722c
c4a10995174bcb09c0d8acb69c01c0a3228665c2
6148 F20101123_AABTUC vanalstyne_r_Page_62thm.jpg
85b1317cb539a89b8b0bdc7e36e49adb
06a8b7743e113a04761ed27920b2e103bceb807e
106326 F20101123_AABTFK vanalstyne_r_Page_25.jp2
cbc2527550dd6915e42729139edd5e6b
183ad9245044a30b68ec2f523e053c3a90b22a18
1790 F20101123_AABTPE vanalstyne_r_Page_63.txt
554178eed218e534960b9e54445f8f77
9fa42bfbb1b59c9bd4e43770187c58744169eab9
33271 F20101123_AABTKH vanalstyne_r_Page_08.pro
10951dff74540dfe9f249d0b9ce55271
11726c6a40edecaf1dbe0b2034fc58c6bc278f42
21063 F20101123_AABTUD vanalstyne_r_Page_63.QC.jpg
747f929c1702706ce31d797fa464f7a5
7e7d4747d56c059ffd71e6dd4b56a362dda0a44a
111093 F20101123_AABTFL vanalstyne_r_Page_26.jp2
d209875ec8cd6923f36b0687277bb097
8f058a3cee72c9389449653b9cc8edf7b1ff43a5
1878 F20101123_AABTPF vanalstyne_r_Page_64.txt
d4891010d46e42b17503744e3df8a685
39dcdbe31027dc8e7f4075d765f185ecc63aba34
37297 F20101123_AABTKI vanalstyne_r_Page_09.pro
df335cc16546dcf43e7686313819564e
2d82a53c2d93a349729dfe22cd63a189e10c4697
5824 F20101123_AABTUE vanalstyne_r_Page_63thm.jpg
8b5ff6ad8af2b7a6f1c0b5fe5e76c5be
487d28f76234b4b21607153b689e143e702f5223
107383 F20101123_AABTFM vanalstyne_r_Page_27.jp2
fae1a5b331556d87fcf2173871f9aae9
cbcb6532027b453288a629c59df43bf35d81da70
1795 F20101123_AABTPG vanalstyne_r_Page_65.txt
1f0e0f57e73d3a2cf279abb32e5cbd41
b995afe086a41a6c71fd7cf3e87cf081c7556a75
42193 F20101123_AABTKJ vanalstyne_r_Page_10.pro
2bad2e216892d4753f5f8f1c62f611a6
1784e51e3f8372dedb10d4b79a005ca3e2e97034
22753 F20101123_AABTUF vanalstyne_r_Page_64.QC.jpg
58c66e13272dbcf4c2592f4e6150ea08
9c3d3bba286a2a221b19193f2f9ef92320af28b9
112169 F20101123_AABTFN vanalstyne_r_Page_28.jp2
51d19606b4ff9f0d24ceefac43745528
8ff28444f92c04a83e875f04b4a0f4234971d32a
1434 F20101123_AABTPH vanalstyne_r_Page_66.txt
ec1cc6e2235bcf5dcbd65e2d570ca7a7
0709081da3ac1334a522101dd26dfe8c2dc89cba
43311 F20101123_AABTKK vanalstyne_r_Page_11.pro
708591238efa25329a18b657e23de99d
bee576c77e26b8bc8bec5e190ff5ffc0b0cba1d4
6450 F20101123_AABTUG vanalstyne_r_Page_64thm.jpg
36d061bbdcb30226403673d110c462f1
8d7bce30c206287e957995ffa9d43ea73af04d95
105233 F20101123_AABTFO vanalstyne_r_Page_29.jp2
e0598513380596cf58d5fde4ea29326d
72b833452d08f9845d05bcdd2d98dcb6a05a8b16
1230 F20101123_AABTPI vanalstyne_r_Page_67.txt
bdaa7435533a5de0c58acee1291538f1
7418333ca7813e28e27fd6709d0dd765d5df4fc3
49579 F20101123_AABTKL vanalstyne_r_Page_12.pro
e26372a68548b84105b2578109cfde28
e60c3afb0062a61991a449ef903c6301f9fc0ced
109054 F20101123_AABTFP vanalstyne_r_Page_30.jp2
278b4d2795cb3d0b8ebd119e7d3792b7
b07c2bd4a7fdd9272140f37ac24a2e938bbdecb3
1832 F20101123_AABTPJ vanalstyne_r_Page_68.txt
021071f48f3d3e0f859c67222217cd2c
8c239b9fd737e2527f6830a068c5ba383dafc684
45524 F20101123_AABTKM vanalstyne_r_Page_13.pro
33d261fe1801026fe8376e934599e7f8
d8122afb1a6647a36f0dd7c8336c39515cae4278
21829 F20101123_AABTUH vanalstyne_r_Page_65.QC.jpg
dc048b8b690c5190fc67606e5f2cdf86
518112af730699dba755ccbd1fa73056e52be299
110666 F20101123_AABTFQ vanalstyne_r_Page_31.jp2
0d13b2e72bca502a79d2f2351b266867
f90d1f9caf1cb794571fb0532e255f65b4eba4f1
2285 F20101123_AABTPK vanalstyne_r_Page_69.txt
99a9187c5d1f596848a35b127065cb9a
3bd79513a8268399db3393dbdac4e8f88180e28f
40539 F20101123_AABTKN vanalstyne_r_Page_14.pro
1dfb5eb23c730b11500a8211db1f70df
8878e78cc18270401f343df55aaea80d3ba1010d
17941 F20101123_AABTUI vanalstyne_r_Page_66.QC.jpg
79d9b553cd096ad2893624cb8d4203d4
382f66ff079e26679d94e976d59e0e5e88f21b3b
31072 F20101123_AABTFR vanalstyne_r_Page_32.jp2
5142159371f71fc272f120e1433a6b04
94586741411a15731f40dc202e466aee753bc93a
2406 F20101123_AABTPL vanalstyne_r_Page_70.txt
d6a624d780a1b8e8f137c9f346b428da
b5bca389ccefb245e040dacde5f9494d22a8c98e
52020 F20101123_AABTKO vanalstyne_r_Page_15.pro
fb3996205c45eed4ef5554ba299f6395
05f2f165826b741ed46a1fe6060f306fbf3e72a2
5157 F20101123_AABTUJ vanalstyne_r_Page_66thm.jpg
c485ab92db6f7a8fafe39a26d8e0200c
88ee089c180aef191700403656ad27a95721f664
105206 F20101123_AABTFS vanalstyne_r_Page_34.jp2
d455dc38b6b1a905adeae8ae494c6c0f
38dabc9971860d7af7c36553da752ffaf1264040
2441 F20101123_AABTPM vanalstyne_r_Page_71.txt
a8ebc27b9e1f3c978e2fb5c00616555c
5dbc2312150c295e6b89e71cc73831925728e238
49804 F20101123_AABTKP vanalstyne_r_Page_16.pro
10e678384589c35bf5d88999601c9c7c
6c0d4da386368748aeac6c78a2eabc450de4a4dc
12506 F20101123_AABTUK vanalstyne_r_Page_67.QC.jpg
02dec60bb145d3154f65d62e842a5491
45df7fc97282ff375b2a7be3031a2f98b52915d6
107282 F20101123_AABTFT vanalstyne_r_Page_35.jp2
5901d34f9f4030a81a50d70ea9c19d7d
b127e7d9e28de1290300b33e38a16f4ef0ae21a1
1343 F20101123_AABTPN vanalstyne_r_Page_72.txt
d2018890071c5ad9a13393b0223c4ff0
f32f184c9c3dc458327d53968cc76d86d9ee9fae
51364 F20101123_AABTKQ vanalstyne_r_Page_17.pro
99d82e900c66216a538f27a029b278a4
b35746d5b38a562d5e3bb86096cf8e52580fd268
3508 F20101123_AABTUL vanalstyne_r_Page_67thm.jpg
39f62f5208ce0a3dccac85a54eaadb1f
b1ae6001d99611fbd6e627f3e29019244d9029a6
106404 F20101123_AABTFU vanalstyne_r_Page_36.jp2
8b3362bd7c568c1ea927eb64eb58dbb8
c4512f8cccc63bed2054de361dacec84446a2dcc
1757 F20101123_AABTPO vanalstyne_r_Page_73.txt
a6664a2fccb78132ce99674ab9929e4b
1112ea9b08d45e5507eb7803a5557d7736e2533b
49467 F20101123_AABTKR vanalstyne_r_Page_18.pro
2ef2a1b9ed37b71b559b410bf5cdff43
1d235b322aff16d416ecf3599edbfdaf4ff40152
19735 F20101123_AABTUM vanalstyne_r_Page_68.QC.jpg
bb83adc5d320449053efc1b6bfde8741
ec0e63a8738de0802a409b4ba87d6701b68ca4bb
108646 F20101123_AABTFV vanalstyne_r_Page_37.jp2
ad8198c38288de6c498b02ceac6a4639
b0a30ce819adb4e16bed2e82d1cfee122a044448
671 F20101123_AABTPP vanalstyne_r_Page_74.txt
ac7fce6ce161965c1487bc1d29075c05
16551c8c916ef8f585a376da99ff0ba06209efaf
48912 F20101123_AABTKS vanalstyne_r_Page_19.pro
3c745c036a1559863bafc85cf5633484
8d2e49c1a33ec064d6ee0eb17ade5d04702f5cda
5464 F20101123_AABTUN vanalstyne_r_Page_68thm.jpg
c913d111858daf4a77071653c2ae5f55
d12d2c521eb2722b88e301cb520c2712b34d99a4
108152 F20101123_AABTFW vanalstyne_r_Page_38.jp2
ce5cce369f39ab469f4c35f7540ea60f
438d614aff23e468e96d8649a93ed32aa5affe79
668548 F20101123_AABTPQ vanalstyne_r.pdf
7085399a157b2f15fdccd2badfe5aaeb
4a5d70a506e953f29a690a0677b213c9105958e5
85362 F20101123_AABTAY vanalstyne_r_Page_33.jp2
e61b482de82a5fd3020be7c9f9b86cb3
637f2d9b37ed34d90ba095f784c40e29f0b79889
50353 F20101123_AABTKT vanalstyne_r_Page_20.pro
cab05c98e8b0dbd691cd757f3cfe33c2
481f43d534cdcd379b6aec1200b275a4dedd743a
23402 F20101123_AABTUO vanalstyne_r_Page_69.QC.jpg
85ce8e25b7453227bdb7483eb4a16fc2
f33e565d0b604a096d790a1845e4153440498983
7592 F20101123_AABTPR vanalstyne_r_Page_01.QC.jpg
bfdc04ffb2721211c6ba1ac6284de906
a0ddf647215696c9a92e8ba0f26a8f16b3eaa4b0
5985 F20101123_AABTAZ vanalstyne_r_Page_53thm.jpg
7b470597630a75676c250592c8854aad
2ac6c30a37ac785a7fb3a913401fa76df81a96d3
49524 F20101123_AABTKU vanalstyne_r_Page_21.pro
d0e646162b4db5b49d4124c843a22508
9e7f40ff88896476f493f81784cec85eb9b31b29
112983 F20101123_AABTFX vanalstyne_r_Page_39.jp2
a053226708522624217cc613c709dc93
152774a7b1658e6899209451dfce333c6ba9d791
6888 F20101123_AABTUP vanalstyne_r_Page_69thm.jpg
767d6be5eaf88c51555c77ffba9f378c
b22b2bfdd279d9114b75d304a454a0a2088cf74a
2544 F20101123_AABTPS vanalstyne_r_Page_01thm.jpg
d7d591eb6c369a82a8e130ec9b3ad355
2ef85a407eec22add39496b59f99e489b5864076
45628 F20101123_AABTKV vanalstyne_r_Page_22.pro
a73a2cb1275bf5d8239b627c0e192820
9e5bd400c9acab1f4bc80721d44165cde9dcda31
23821 F20101123_AABTUQ vanalstyne_r_Page_70.QC.jpg
17d127632a21a22f5316fe5080ff7a5f
eed27100ed08530dddaeeb0bbb1c66331b2ca182
3332 F20101123_AABTPT vanalstyne_r_Page_02.QC.jpg
d1998c2e48ab7d7194b7caf6f1ca56ed
2523386257b08a35fe93033303886cae08da1997
47849 F20101123_AABTKW vanalstyne_r_Page_23.pro
48a1e77f75f8d57e19b1c4bd68e11967
ca0671e7030e06e2089d5eca3bb2eb8267af5e3b
69975 F20101123_AABTDA vanalstyne_r_Page_36.jpg
54dfa17d3fde3e94fb26772824edca64
d71cf281c4847be189ecc50a6621717b7b0f70db
109274 F20101123_AABTFY vanalstyne_r_Page_40.jp2
ace299bc5de4f962a891e128160c84b4
aa4dc7fd824ad03dae42bb8a3426e63021aa7036
6787 F20101123_AABTUR vanalstyne_r_Page_70thm.jpg
adc26eab7392bec581ee24541aac1af9
77d19562ef4d4a6336e0d0bc2545f42eac84fedc
1384 F20101123_AABTPU vanalstyne_r_Page_02thm.jpg
0f41efffe6ad17a375ef6d6e0a562266
e2fc78b09167a6795731039b45d3422be055a466
51620 F20101123_AABTKX vanalstyne_r_Page_24.pro
930e62819a200c1c087456e9de3ec12f
81e923162c6011b6c317ea405e258921508d4f68
71641 F20101123_AABTDB vanalstyne_r_Page_37.jpg
ea7073f53ba8a13bf40dc95aa404feb6
13462353f27e3ef2e859f1f01aed4331806c4f74
111253 F20101123_AABTFZ vanalstyne_r_Page_41.jp2
68b7a65774438faeef646c14fc0070fd
c7c730c1082c0746432ad9fcb83b76121b59cbcc
25505 F20101123_AABTUS vanalstyne_r_Page_71.QC.jpg
fdedbe65cb975173f21c958a8399e808
e43351831c14998c0999b06d3570d3026a24a172
3538 F20101123_AABTPV vanalstyne_r_Page_03.QC.jpg
3a1c395186bd2398f76ecb97a619666f
334ed113e850c7573998e0639aad2611ec6ab1af
49707 F20101123_AABTKY vanalstyne_r_Page_25.pro
ce80e797e3a6003576188bdddd77e165
54f52fbc21b8fb904c8d2f68f6edd563820ff256
72514 F20101123_AABTDC vanalstyne_r_Page_38.jpg
3653b6ef06a4085de34cc5c1bfbea011
b2e508628ad18451ae53485811663af63777b328
6773 F20101123_AABTUT vanalstyne_r_Page_71thm.jpg
b04f87f26fe6e1c854cd16b69e7a06b5
b48a71b4a3ce270fe6d4dc7db0307ab01873fc20
1686 F20101123_AABTPW vanalstyne_r_Page_03thm.jpg
a4c12dab261b8246c541103ce33e4596
53276e10b1df2405d12372021cd9348fc08b8039
F20101123_AABTIA vanalstyne_r_Page_22.tif
ade45747dff5fc5affb5827f7375df7e
4b70db42683177fa2c9c0e11641cabe31d8034f5
50437 F20101123_AABTKZ vanalstyne_r_Page_26.pro
b695906cfa00a7bf304a590fc1a8de5b
972633050fed2579af77c11217490a6796570051
74719 F20101123_AABTDD vanalstyne_r_Page_39.jpg
9a7132316cbd8d7d1e910d3a86f19bcb
10286e7a3df473be35fe7be0cd62c82e20dd5d3f
14096 F20101123_AABTUU vanalstyne_r_Page_72.QC.jpg
51b2bb752a2f9acb660b3513198146dd
ec4be4495ab44e214ebfde9c53066e0b202eecba
20405 F20101123_AABTPX vanalstyne_r_Page_04.QC.jpg
ddb9c4db0d6e3230f5878c660f86920d
52960b8486218a401ff7f378097c2a1bab4814c0
F20101123_AABTIB vanalstyne_r_Page_23.tif
0fbb9a0d2c52621ebdf36d46a9d70552
c137517794640323154546fabbbf4058e028c6c8
72178 F20101123_AABTDE vanalstyne_r_Page_40.jpg
c32ca78b44506da8cb004974d2ba1349
85e29fd9f54ad085dcaacf56c20330871c8727f1
4186 F20101123_AABTUV vanalstyne_r_Page_72thm.jpg
0a9b705b17d33dbc10b5974d395d5a13
42d0603fbca703f8c42a93d280de3b3b1d07b09f
5984 F20101123_AABTPY vanalstyne_r_Page_04thm.jpg
885af9aa8fa8fbc07820a5fdf0bfaddc
3254756040a70f31b3bf0a161a1354752ad1b346
F20101123_AABTIC vanalstyne_r_Page_24.tif
64b31d4792b1dce968f450aadab76699
f20af7d9ed24224a5f2a6947bdf8cd579fbf7c59
74694 F20101123_AABTDF vanalstyne_r_Page_41.jpg
74bae33a4f8bd0806d4b1df0b1e19ca0
c29da3f377d22e5549cbda2f668fa511426a73ab
21248 F20101123_AABTUW vanalstyne_r_Page_73.QC.jpg
df20fe1739b910e2e0674a7c051ce0e4
1b22fefd16548a5ca77b27cb9951a015ec161e07
8401 F20101123_AABTPZ vanalstyne_r_Page_05.QC.jpg
c1d7a04b369d7ff0f8ccad226ab10747
ea56a2ada54be58485d7d683676e2788e111211d
F20101123_AABTID vanalstyne_r_Page_25.tif
aa5b0f3288312ef89539ea0409750ce4
acf9cace9ca10f7b40dc388f2713c161c72d5ec6
71884 F20101123_AABTDG vanalstyne_r_Page_42.jpg
ec63e76ebd022f0df69b37e0aef3ba39
12ec6f780cec09bb02d60bc0f9afd6f29f2b7087
1954 F20101123_AABTNA vanalstyne_r_Page_07.txt
193c54e70526aef751d34c7e044311ef
2356170efb38d9b333227e92d9215b36a977f669
5855 F20101123_AABTUX vanalstyne_r_Page_73thm.jpg
4aa693db049bbbb25dd71cecaac383bf
4e0b7d61b3aae62700392323b7383d400a9242a8
F20101123_AABTIE vanalstyne_r_Page_26.tif
024bd5a603c574436992078885700d38
9c06c32da86937f1e6250422dcd6ea3df6d35557
73090 F20101123_AABTDH vanalstyne_r_Page_43.jpg
ca62d98cad4b0266f15177e1aab7a1d7
7243577ba66f0e0790cef5ff0e33af3a6624ecb6
1380 F20101123_AABTNB vanalstyne_r_Page_08.txt
8a867f08e6e549b0f7941cc00e206827
f7bf145c0a45f75b70e63e11e2a64b4fbc5ad963
9554 F20101123_AABTUY vanalstyne_r_Page_74.QC.jpg
e93640872fc1a6f411ae617fbe395a13
1fbff6795d2c12164ee00b9c778cc983f4ae5d3c
19703 F20101123_AABTSA vanalstyne_r_Page_33.QC.jpg
478b2dac0bae0744ac4e8f0a5612cf04
a940983102ded9d0171a0c054791c461bba71246
F20101123_AABTIF vanalstyne_r_Page_27.tif
7ecc323fc2f085cf94def82434065f7a
adbea491b7ec13eddaf37c28dc405a3b4ca0ab5a
72772 F20101123_AABTDI vanalstyne_r_Page_45.jpg
efc0e75ce91d2e7f8bbb9e72f857ddd0
1f0da6abe68066c2dae03db1637a2361c0929158
1634 F20101123_AABTNC vanalstyne_r_Page_09.txt
09c0f01e8728d6c3525b5da024592717
935128659500e1d56d9a73e7ef20d80544438f05
2925 F20101123_AABTUZ vanalstyne_r_Page_74thm.jpg
0357c2e93c4807f93e5a8c3b9abc127a
07edd45bcbc8205bd6ec7900306ee2069433ac26
5555 F20101123_AABTSB vanalstyne_r_Page_33thm.jpg
8b24fe6f8f30ff9b43724dda5520a4b5
626b4b91d4107ef4ae3a0760e7cc45e782b70298
F20101123_AABTIG vanalstyne_r_Page_28.tif
0fc8fd0a2f0f10e2e3e41c07939c1eca
5f32ed0050c2b11bc4d99a489fc41145c4a8b252
71416 F20101123_AABTDJ vanalstyne_r_Page_46.jpg
44544cb852853c9cb7d707fbf7d2675a
818bf9d92d3e205931b99c84f902e13159498313
1674 F20101123_AABTND vanalstyne_r_Page_10.txt
0932b2533b32dc4e835f76d8c5ccfe8a
7c0f3ad5c705978a88b75cd336debd21a26371d8
22947 F20101123_AABTSC vanalstyne_r_Page_34.QC.jpg
db026e02635ff82cbe9d50f061ab6cdf
ea5b7c82cab56e2eaf64e59c2b31fdae8772ed87
F20101123_AABTIH vanalstyne_r_Page_29.tif
1d473fbeee49e1e4cab7268cdda7709d
2b1bb8b472cea28d771a537bef612388f2c47400
38467 F20101123_AABTDK vanalstyne_r_Page_47.jpg
c8d3fa33e78cd55dc2534f26166738c2
0e0c48a9ba0833c2f5371c6dde580ecd27037694
F20101123_AABTNE vanalstyne_r_Page_11.txt
e1a1505dd0266706698df8a31fd49cdf
204aacb0b386f36e0eb6c1fc3873723a33efba89
6311 F20101123_AABTSD vanalstyne_r_Page_34thm.jpg
5faac773f543e70e57a5e8de5138e0ab
b661f39973db9e0c327649c22e75ec525bc80dcf
F20101123_AABTII vanalstyne_r_Page_30.tif
4798be6dfee1d0aa428c36e0a18bb99e
06591aa79763d71dc205c02f043a095026ce4e71
80859 F20101123_AABTDL vanalstyne_r_Page_48.jpg
4fb7315fe93d8938f2ecc5668fc31cdd
907b677b5ed198345c8ed9245d8e4d7b65610cf2
1947 F20101123_AABTNF vanalstyne_r_Page_12.txt
2108852b0b6271928d193ea224d23212
8ab1b69a01d246ba8aac10c6ad359985dfee9808
23555 F20101123_AABTSE vanalstyne_r_Page_35.QC.jpg
95b53fb76e1360ecbc03156146b24d3c
38e4198ba366e8ae3c12af218ff1e70fabdef424
F20101123_AABTIJ vanalstyne_r_Page_31.tif
78fb804f3dcff7f63cb3731129bb5883
4e28c390676ec877467518245ead4fb4b2756f05
79171 F20101123_AABTDM vanalstyne_r_Page_49.jpg
2a40b0787d625eaf703c30d3152b6013
aef874c60b05e77235491cb0503473bf29b4cd1e
1806 F20101123_AABTNG vanalstyne_r_Page_13.txt
58ce144c78a2c88c4209baaf54b51ef9
7571f8c524752ce0c7e6230db8db4ecf610c445c
F20101123_AABTIK vanalstyne_r_Page_32.tif
7360c515820c13b21740b93516fdae7a
5a826b664d5f42c8dd9ec3ead6481cfa016a4e8d
45113 F20101123_AABTDN vanalstyne_r_Page_50.jpg
aa50b964a72c83b4969b0156a98cac6a
ed573fe072e910d9645469e3fc274ef479d0f88e
1697 F20101123_AABTNH vanalstyne_r_Page_14.txt
10eb3483be2fdd9e6a8d98e3b7ae9654
d5e79037aa11ba85b85ce80692eea8f267f26bc1
6452 F20101123_AABTSF vanalstyne_r_Page_35thm.jpg
040b1a23fa55ff23a34e80697355318c
b3eab33fd6f4ac8d75e07d5f1e9aa28b0fe3b2dd
F20101123_AABTIL vanalstyne_r_Page_33.tif
02ee4a02e372a6896d476fb170f22b26
d6e5324733d44e3c199997124d93e66945875f93
68467 F20101123_AABTDO vanalstyne_r_Page_51.jpg
b92dbebef163ecadbe194e5630da44bb
14d16494384b654a9204c13abb1a96175febbeeb
2077 F20101123_AABTNI vanalstyne_r_Page_15.txt
3c4ac0078a461a08c43224404b3c5d0c
5618224d2196688e508f0f0876ac848b5f0584ee
F20101123_AABTSG vanalstyne_r_Page_36thm.jpg
4a2c970033629f3ddf6d8e21ec6f92e2
fb1f3bedad208066bec44f37f0abd15afffa8425
F20101123_AABTIM vanalstyne_r_Page_34.tif
87c9602b4d6aa08712737519f479f025
15396895207e007a08e3b52fa43771a934c42e37
37985 F20101123_AABTDP vanalstyne_r_Page_52.jpg
a99fe949ded0a37af1c7d9fdd70b80f8
31a519debe0005515431751c0bc62af434141d29
1991 F20101123_AABTNJ vanalstyne_r_Page_16.txt
9c16f9aa3db0c8ca2c10f3cf25a2a3f7
ace432f9f1b21f477120ac3f852e6155f28fdfeb
24131 F20101123_AABTSH vanalstyne_r_Page_37.QC.jpg
b6f85799755b312c11f57ea60ada69dd
191272016bc505a8b371aebcf1cb4ae82701bc26
F20101123_AABTIN vanalstyne_r_Page_35.tif
ebe0ae5f3048041beab384b65fe7f6f9
170e1e943568cb5fa593571262b13f15e56c7283
63646 F20101123_AABTDQ vanalstyne_r_Page_53.jpg
a5292f42c8a346842bbbe9edaaa3709f
1a3a711d5b77626703ea901784febb17ac1099f5
2014 F20101123_AABTNK vanalstyne_r_Page_17.txt
0544845ed450b8729d12662ca17663be
5d2b32fa1c740f3b38c0dd6b8550061d12899209
6722 F20101123_AABTSI vanalstyne_r_Page_37thm.jpg
3a75adadd7bf4ca3b82bb2dd011a7241
1e2f8fa5da99452b674120c27618deec5cbb233f
F20101123_AABTIO vanalstyne_r_Page_36.tif
a49cd8fb04863ba3d3f4d7d85a463b91
c360762208d6193beae40a65c5596fb7fa867dc8
74053 F20101123_AABTDR vanalstyne_r_Page_54.jpg
d19e2143808be2050a6fbf5614c74303
9ad5bb52bb6aa15ea4030013dd9ec55a4c410da5
1967 F20101123_AABTNL vanalstyne_r_Page_18.txt
dfa7cc029f6eb294bc2d94e07c1e9565
d0cdceb5b275158bbcd2bab4562f1fa4e1999fc5
23740 F20101123_AABTSJ vanalstyne_r_Page_38.QC.jpg
ad270fc0d1a45ecbe8a8b56af60a9fb4
8c25f802d5cce138c46a3a49315301e36e33fa96
F20101123_AABTIP vanalstyne_r_Page_37.tif
926441b9a5b506c55a07797569786fa2
17c37f1b860d6eaaabe418b2f3b520eb33cb8a4b
69034 F20101123_AABTDS vanalstyne_r_Page_55.jpg
17e64060d035a6b6690a72a832ffa557
3f53385ddbef61f927bbc6f8b3edf3ddd57f0857
1940 F20101123_AABTNM vanalstyne_r_Page_19.txt
af9ea395ce38bedea5cfe76195ec197f
98fc4b6d906a50107c0a1dd445cc8271a21c27a4



PAGE 1

EFFECTS OF DIETARY ALUMINUM FR OM WATER TREATMENT RESIDUALS ON PHOSPHORUS STATUS AND BONE DENSITY IN GROWING LAMBS By RACHEL VAN ALSTYNE 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 2005

PAGE 2

Copyright 2005 by Rachel Van Alstyne

PAGE 3

Dedicated to family and friends who stood by me through my educational journey.

PAGE 4

ACKNOWLEDGMENTS The author wishes to extend sincere gratitude to Dr. Lee McDowell, Dr. George OConnor, and Dr. Lokenga Badinga for their service on her graduate committee. Thanks are extended to Dr. McDowell for his support, patience, and encouragement throughout this endeavor. Thanks are extended to Dr. OConnor for his professional wisdom and support regarding soil science. Thanks are extended Dr. Badinga for his valuable insight and suggestions. The author would like to thank Dr. Paul Davis for his assistance throughout the trial and writing processes. His hard work, loyalty, love, and support as a friend, partner, and colleague have been paramount to the success of the author during her life while attending the University of Florida, in the publication of this thesis, and in the life they seek together in the future. Much appreciation is extended to Nancy Wilkinson, Jan Kivipelto, and Dr. Maria Silveira for aid in laboratory analyses and data interpretation. Their support, time, and assistance have been priceless. Thanks are given to Dr. Lori Warren, Steve Vargas, Jessica Scott, Carlos Alosilla, Kathy Arriola, Eric Fugisaki, Tom Crawford, Jos Aparicio, and Luis Echevarria, for their hard work and support during the trial. Additionally, the author would like to thank her best friend and roommate, Karen Fratangelo, for her support and kind regards during stressful times. Karen and the author have been friends since the 6th grade and have been able to stay close and lean on each other. Karen aided in diapering sheep, lended an ear, and shared a kind heart during hard iv

PAGE 5

times. Though it may seem odd, the author would like to give her kind regards not only to the humans who aided in her success, but also to the lambs that will always have a soft spot in her heart. Last but not least, the author would like to thank her parents, brother, and canine companion Vixen for their love and support throughout her education and throughout life. She would never have had the perseverance and confidence that she does without their love, loyalty, support, encouragement and respect. v

PAGE 6

TABLE OF CONTENTS page ACKNOWLEDGMENTS.................................................................................................iv LIST OF TABLES...........................................................................................................viii ABSTRACT.......................................................................................................................ix CHAPTER 1 INTRODUCTION........................................................................................................1 2 REVIEW OF LITERATURE.......................................................................................4 Historical Significance of Phosphorus..........................................................................4 Requirements................................................................................................................5 Phosphorus Deficiencies...............................................................................................6 Phosphorus Metabolism and Transport........................................................................8 Aluminum and Phosphorus Interactions.....................................................................10 Pollution and Phosphorus Application to Land..........................................................14 Regulations.................................................................................................................16 WTR and Environmental Uses...................................................................................19 3 EFFECT OF ALUMINUM-WATER TREATMENT RESIDUALS ON PERFORMANCE AND MINERAL STATUS OF FEEDER LAMBS.....................23 Introduction.................................................................................................................23 Materials and Methods...............................................................................................24 Animals, Diets, and Management.......................................................................24 Sample Collection, Preparation, and Analyses....................................................25 Statistical Analysis..............................................................................................26 Results.........................................................................................................................27 Discussion...................................................................................................................29 Summary and Conclusions.........................................................................................36 Implications................................................................................................................37 vi

PAGE 7

4 EFFECT OF DIETARY ALUMINUM FROM WATER TREATMENT RESIDUALS ON BONE DENSITY AND BONE MINERAL CONTENT OF FEEDER LAMBS......................................................................................................43 Introduction.................................................................................................................43 Materials and Methods...............................................................................................44 Animals, Diets, and Management.......................................................................44 Statistical Analysis..............................................................................................47 Results.........................................................................................................................47 Radiograph BMC.................................................................................................47 Bone Density via Specific Gravity......................................................................47 Bone Mineral Analyses.......................................................................................47 Discussion...................................................................................................................48 Summary and Conclusions.........................................................................................49 Implications................................................................................................................50 5 SUMMARY AND CONCLUSIONS.........................................................................53 APPENDIX: TABLE DATA.............................................................................................57 LITERATURE CITED......................................................................................................58 BIOGRAPHICAL SKETCH.............................................................................................63 vii

PAGE 8

LIST OF TABLES Table Page 3-1 Diet composition (as-fed) and analyses for average (n=18) concentrations for macroand micro-elements for treatments...............................................................38 3-2 Effects of dietary Al concentration and source on BW of feeder lambs .................39 3-3 Effect of dietary Al concentration and source on feed intake of feeder lambs .......39 3-4 Effect of dietary Al concentration and source on plasma P of feeder lambs ..........40 3-5 Tissue mineral composition resulting from experimental diets ..............................41 4-1 Diet composition (as-fed) and analyses for average (n=18) concentrations for macroand micro-elements for treatments...............................................................51 4-2 Effect of dietary Al concentration and source on bone density of feeder lambs as determined by radiography .....................................................................................52 4-3 Effect of dietary Al on dry fat-free bone mineral concentrations of Ca, P and Mg for experimental diets ..............................................................................................52 A-1 Effect of dietary Al concentration and source on ADG of feeder lambs ................57 viii

PAGE 9

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 EFFECTS OF DIETARY ALUMINUM FROM WATER TREATMENT RESIDUALS ON PHOSPHORUS STATUS AND BONE DENSITY IN GROWING LAMBS By Rachel Van Alstyne August 2005 Chair: Lee Russell McDowell Major Department: Animal Sciences Experiments using growing feeder lambs were conducted to gather data on 1) the safety of a Al-water treatment residual (WTR) ingested in amounts to provide between 2,000 and 8,000 ppm Al, and 2) the bioavailability of Al in WTR when compared to a control (910 ppm Al from sand) and a diet containing a known bioavailable form of Al from AlCl3. The study was conducted to examine changes in performance (ADG, BW, and feed intake), tissue mineral concentrations, plasma P concentrations, bone mineral content (BMC), bone density, and apparent P absorption. At experimental termination, samples of brain, liver, kidney, heart, and bone were collected and analyzed for concentrations of Ca, P, Mg, Cu, Fe, Mn, Se, and Zn. Thirty-two wether and ten female lambs were assigned to six dietary treatments: 1) control (10% sand), 2) (9.7% sand and 0.3% AlCl3), 3) (2.5% WTR and 7.5% sand), 4) (5% WTR and 5% sand), 5) (10% WTR and 0% sand), and 6) (10% WTR, 0% sand, plus double the quantities of the mineral-vitamin premix, ix

PAGE 10

and 1.29% dicalcium phosphate). Treatments 1-5 contained P at 0.25% and concentrations of Al were 910, 2000, 4000, 8000 and 8000 ppm, respectively for the six diets. Compared to the control, ADG, BW, and intakes were unaffected by dietary levels of WTR (P > 0.05); however lambs fed 2,000 ppm Al from AlCl3 had reduced body weights and lower ADG (P < 0.05). The control, most often, had the highest plasma P concentrations and the WTR treatments generally had higher P concentrations than lambs given AlCl3. During wk 6, plasma P concentrations declined for all animals but steadily increased thereafter. Kidney P differed; control lambs had larger deposits of P than lambs given 8,000 ppm Al from WTR (P < 0.05). Iron deposits were highest in livers from lambs fed 8,000 ppm Al from WTR and lowest in the controls (P < 0.05). Brain Al was highest for animals receiving 2,000 ppm Al from AlCl3 and lowest for lambs given 2,000 ppm Al from WTR (P < 0.05). Brain Al concentrations increased when Al from WTR was given in amounts above 2,000 ppm. Apparent P absorption did not differ among WTR treatments and the control (range from 11 to 32 %), but lambs fed 2,000 Al via AlCl3 had a negative (-13%) apparent absorption of P. Values of BMC and bone density did not vary with treatments; this is likely due to the short duration of the study. This study found no evidence of health related defects because of the administration of the WTR. The Al as AlCl3 was more bioavailable with regard to plasma P levels and performance, than Al via WTR; animals which were given the AlCl3 were negatively affected. x

PAGE 11

CHAPTER 1 INTRODUCTION Manure transportation for refuse is costly. This results in the primary method for animal waste disposal being application nearby land. Repeated long-term manure land application leads to accumulation of phosphorus (P) (Novak and Watts, 2004). In the United States, the livestock industry produces 500 million tons of manure each year (Lorentzen, 2004) and many coastal soils have already become saturated with P (Novak and Watts, 2004). The majority of the P produced as animal waste is not adequately used for plant uptake and much of the soil used by large industrial agriculture companies has reached its maximum capacity for P adsorption (Novak and Watts, 2004). When manure is applied to the land and the P remains stagnant on the upper crust of the soil bed it may be washed away with heavy rains (Haustein et al., 2000; Federal Registar, 2004). Phosphorus lost in leaching and runoff can lead to eutrophication, causing the overgrowth of algae, and decreasing the survival of aquatic plants and animals (Novak and Watts, 2004). These algae lead to a reduction in the oxygen levels within the water and result in an overgrowth of anaerobic bacteria that generate toxins such as Pfissteria which may result in death, rashes, respiratory illness, and memory loss in people and animals (Haustein et al., 2000). The death and decomposition of aquatic plants can lead to depressed aquatic oxygen levels, resulting in fish mortalities. Aluminum (Al) applied to the land has been shown to reduce the soluble P concentrations from animal waste (OConnor et al., 2002). Aluminum salts have been 1

PAGE 12

2 used to help minimize the amount of P released from animal feces. The chemical reaction which occurs between the Al from salts and the P in the manure, result in a decrease in P loss. This method has proved to be effective but it is costly (OConnor et al., 2002). The reduction of available P levels from animal waste products could result in significant decreases in leaching and runoff, lessening contamination of water supplies. Studies conducted at the University of Florida suggest that water treatment residuals (WTR), especially those containing Al, increase the soils capacity to retain P. Water treatment residuals bind P, lessening its availability and decreasing water pollution caused by runoff (Elliott et al., 2002). Water treatment residuals are derived from the water purification processes and can vary in their mineral content and P absorption capacities, depending on the chemical used by the water treatment facility and the age or dryness of the WTR. Thus, WTR can be high in Al, Fe or Ca oxides and upon drying or aging become safe for land application (OConnor et al., 2001; Dayton et al, 2003). The WTR used throughout this experiment (including references, unless otherwise stated) are high in Al-oxide from a source known for its high P sobbing capacity and will be referenced as WTR. Water treatment residuals are the solid sediments that result after raw water is coagulated, leaving behind amorphous Al oxides (Basta et al., 2000; Dayton and Basta, 2001). These WTR contain amorphous solids that vary in size and shape. Most WTR look like, and have the texture of a dark soil, but have little or no nutritive value. However, WTR usually contain 4 to 8% nonavailable Al. In general, WTR are discarded in landfills or in waterways, but both methods of disposal are costly and may increase the price of drinking water (Novak et al., 2004).

PAGE 13

3 Application of WTR to the land may result in one solution for animal waste discard, while eliminating the burden and expense of WTR disposal. The chemical mixture of soil and WTR has been proven to increase the retention value of soil P by several fold (Novak et al., 2004). One concern posed is that WTR contains high amounts of Al leading to contamination and ecological risk for grazing animals, wildlife, surrounding floriculture and water systems (USEPA, 2003). High levels of Al can adversely affect P utilization and bone deposition. Aluminum toxicity is often observed as a P deficiency resulting in bone density impairment (Valdivia, 1977). Toxicity is primarily linked to the degree of Al bioavailability. In WTR, the bioavailability of Al varies, but is generally low (OConnor et al., 2002). During grazing, ruminants naturally consume up to 10% to 15% of their total dry matter (DM) intake as soil (Field and Purves, 1964), and soil can contain as much as 10% Al (Valdivia, 1977). Research with livestock at the University of Florida demonstrated that increases in dietary Al decreased voluntary feed intake, and feed efficiency, depressed P serum concentrations, and depressed growth and gains (Valdivia, 1977; Rosa et al. 1982). In this research Al-WTR were directly fed to sheep to simulate grazing-like conditions and soil consumption to emulate an ingestion of soil material in amounts of 10% of their diet, for hypothetical assessment of health related affects if inadvertent consumption of WTR was to occur. The following experiments compared the bioavailability of Al from WTR to an available source of Al, in AlCl3. The main focus will be on the effects of these Al sources on P status in sheep.

PAGE 14

CHAPTER 2 REVIEW OF LITERATURE Historical Significance of Phosphorus Phosphorus (P) and calium (Ca) are the two primary minerals that constitute bone matter and are actively involved in bone development. Together, Ca and P are the most abundant minerals in an animals body (Miller, 1983). Eighty to 85% of an animals P is found in the bones and teeth. Combined, Ca and P make up 70% of the minerals found in the body (McDowell, 2003). The essentiality of P in bone development has been known since 1769, when bone ash was analyzed, and P was found to be a primary component of bone material (McDowell, 2003). Much of the early research on P was instituted in areas of South Africa where deficiencies had become a growing concern. Low P diets had been linked to lamsiekte and botulism in much of the grazing livestock throughout the continent of Africa. Clinical signs which appeared included: bone chewing, depressed growth, failures in reproduction, and reduced feed intake. Chewing the bones of dead carcasses is the most significant indicator of a low P. The P deficiency forces the animal to find any source of P, but the ingestion of the bones can also lead to consumption of Clostridium botulinum and death (McDowell, 2003). In areas of Piaui, Brazil, 20,000 to 30,000 cattle die yearly because of botulism. Today, low P diets and associated diseases are problematic in tropical regions of the world. In Latin America, 73% of all forages evaluated in a feed table publication were P deficient (McDowell, 1997). 4

PAGE 15

5 Requirements Phosphorus makes up 0.12% of the earths crust by volume but is not often found in an uncomplexed form. Phosphorus is extremely reactive and is most commonly found as phosphate in sedimentary rock deposits (McDowell, 2003). Requirements for P vary depending on age, sex, activity, bioavailability of P, protein and energy in the feed, stress, interactions between feed ingredients and nutrients, digestive anatomy and reproduction status of the animal (Miller, 1983). Ruminant animals have a lower requirement for P and Ca than carnivores and omnivores. For strict carnivores such as felines, the requirement for P is 0. 60% and 0.80% for Ca, non-lactating adult humans require 0.70 to 1.25% P and 0.70 to 0.90% Ca while sheep require 0.16 to 0.38% of dietary dry matter (DM) P and from 0.20 to 0.82% of DM Ca (NRC, 1985). The NRC (1985) estimated endogenous loses of P, using a factorial method, to range from 20 mg/kg body weight at maintenance to 30 mg/kg body weight in growing lambs. For proper absorption, P must be present in a bioavailable form. Ruminants, unlike mongastrics, are able to use phytin P from plants and it is considered to be available. Only about one third of P in most plants is available to nonruminants (McDowell, 2003). Incomplete uptake can be linked to an unavailable chemical form of the mineral in the plant, physical barriers in the plant wall, or antagonist elements such as oxalic acid and phytic acid which can bind P, Ca, Fe, Mn, and Zn (McDowell, 1997; 2003). The amount of Ca and P found in feedstuffs varies among sources. The Ca: P ratio in legumes is between 6:1 and 10:1 and is considered to be extremely low in P. Grasses, if mature, are often low in both Ca and P. The values depend on soil conditions and plant species. Alkaline soils are more abundant in trace minerals than acidic and sandy soils. Tropical soils are usually older and acidic, marked by leaching and high environmental

PAGE 16

6 temperature with compromised mineral contents, both in the soil and plants (McDowell, 1997). Seeds and seed by-products are rich in P, whereas animal by-products, tankage, and milled flours are rich in both Ca and P (NRC, 1985). Animals on pasture will develop P deficiencies before those fed high concentrate diets, as grains are high in P (McDowell, 2003). Many factors can influence absorption of minerals such as age, diet, parasites, environmental stresses, disease, and toxic constituents. Growing animals naturally have higher requirements for Ca and P because of bone development. A high protein and energy diet increases the need for both Ca and P, but also increases the ability of the animal to retain these minerals. During the rainy season in areas of South Africa and South America, incidences of P deficiencies are common because more lush forages are being consumed. The increased intake of energy and protein rich grasses increases mineral requirements. Without supplementation, the forages are unable to provide many of the needed minerals in sufficient amounts (McDowell, 1997). Infections and parasites can affect the uptake of P and Ca. Nematodes have been proven to cause demineralization of bone tissues in sheep (Underwood and Suttle, 1999). Stress and activity of the animal will influence mineral needs, those under more stress or those with higher physiological need, including growth, pregnancy, and lactation have the greatest mineral requirements (McDowell, 2003). Phosphorus Deficiencies The most prevalent mineral element deficiency for grazing animals worldwide is lack of P. The requirements for P and, frequently, other minerals are often not met by grazing ruminants, and supplementation is often required. Additionally, certain elements found in low pH tropical soils, such as Fe and Al, can hinder P absorption in the animal.

PAGE 17

7 Deficiencies in P for grazing ruminants have been reported in 46 tropical countries in Latin America, Southeast Asia, and Africa. The soil and forages in these livestock-grazing areas of tropical countries are low in P (McDowell, 2003). Phosphorus deficiency is most often seen in cattle and other grazing ruminants. Young grasses may contain 0.3% P, but mature forages may contain 0.15% P or less (McDowell, 1997; 2003). When dietary P becomes low, an early physiological response is a decline in inorganic plasma P. Normal plasma P levels in ruminants are between 4.5 and 6 mg/100 ml. Levels below 4.5 mg/100 ml in ruminants are considered deficient. A normal P level in ovine whole blood is between 35 to 45 mg and in plasma is between 4 to 9 mg per 100 ml, both of which will vary with age, and sex. Anorexic conditions are first to occur with declines in P, decreasing feed efficiency and slowing energy metabolism in ruminants, which ultimately results in a decline in growth (McDowell, 2003). Dry matter intake was reduced 40% in lambs receiving low-P diets and DM digestibility was less than in animal offered the high-P diets (McDowell, 2003). A significant decline in mineral P concentrations reduces the ability of animals to properly digest fiber, protein and carry out normal metabolic functions (Miller, 1983). Reproductive status may be compromised, primarily in females, which is often the most economically damaging aspect of production. Animals deficient in P have been known to go two to three years without calving (McDowell, 1997). Ruminants deficient in P are listless, with swollen joints, abnormal stance, lameness, and have rough, dry hair coats. Deficiency of P or Ca is similar to that of a deficiency in vitamin D, and lack of any of these nutrients leads to rickets. Clinical signs can include weak bones, which may become curved, enlarged hocks and joints, dragging of hind legs, beaded ribs and

PAGE 18

8 deformed thorax (McDowell, 2003). Bone density decreases and the bone matrix becomes soft and porous. With use of a noninvasive dual photon absorptiometry technique, Williams et al. (1990) determined that dietary levels of 0.12 to 0.13% P lead to bone demineralization in Angus heifers. Prior to bone disruptions, an animal suffers stunted growth, depressed appetite, and weight loss. If the skeletal system is affected, the bones (including: ribs, vertebrae, sternum, and spongy bone material) demineralizes quickly. The last bones to be affected are the long bones and the smaller bones of extremities. When P is deficient, even during normal activities, the bones can bend and fracture, (McDowell, 2003). Phosphorus Metabolism and Transport Calcium and P regulation occurs as a result of the hormones, 1, 25 dihydroxy cholecalciferol, parathyroid hormone and calcitonin. Regulation of normal Ca and P levels depend on bodily excretion, bone deposition, resorption, and intestinal absorption (Miller, 1983). Phosphorus and proper availability of P depends on the Ca to P ratio, of which should be between 1:1 or 2:1 for most monogastic species. However, for ruminants, ratios below 1:1 and over 7:1 will negatively effect growth and feed intake. If Ca and P needs are not met, tetany will occur as the animal withdraws Ca and P from bone in order to maintain normal blood concentrations. The status of vitamin D is important to obtain a desirable Ca:P ratio (McDowell, 1997; 2003). Over time, if Ca and P concentrations are low, bone becomes soft and bone density is impaired. Most of the Ca and P in bone is in the form of calcium phosphate and hydroxyapatite. The exact make up of bone material varies with age, sex, physical activities, and reproductive status, but consistency is seen within species and their stages of life (McDowell, 2003).

PAGE 19

9 Absorption of P in ruminants occurs throughout the intestinal tract, including the rumen, but is optimized in the small intestine. Its uptake occurs through active and passive diffusion and is dependent on the solubility of the membranes that it comes in contact with. Absorption is favored when the mineral is held in solution. Factors which effect uptake of P include: digestive system pH, age, parasites, and other mineral intakes, particularly Ca and Al (McDowell, 1997; 2003). Large amounts of bioavailable Al form insoluble phosphates which bind P, making it unavailable to the animal (McDowell, 2003). Bone undergoes turnover daily and in turn affects the P plasma levels of the animal. Osteoblasts cause new bone formation, while osteoclasts (large multinucleated cells) reabsorb the bone tissue. Most of the nonskeletal portion of P is found in the red blood cells, muscle tissue and nervous system. Much of this P is used to regulate oxygen and hemoglobin in the blood. Status of P in the body can be estimated by plasma or fecal excretion. Feces is the primary pathway for P excretion in ruminants and non-carnivorous animals. Carnivores excrete more P in the urine over that in the feces. In diets low in P, the body naturally conserves P, particularly in herbivores and little to no P is excreted in the urine (McDowell, 2003). Phosphorus is used in almost every metabolic system, including those of ruminal microorganisms, digestion, appetite simulation, feed conversion, fatty acid transport, metabolism of nucleoproteins, maintenance of active cells, enzymes, hormones, and for milk, egg, and muscle synthesis (Miller, 1983; McDowell, 1997; 2003). Evaluation of P status can be determined by the concentration in bone, since the majority of the mineral is in bone. Heifers fed low (0.12%) P diets at had a much lower

PAGE 20

10 cortical bone index, medial lateral wall thickness, breaking load, and total ash than those receiving a 0.20% P diet (Williams et al., 1991b). Bone density of the ribs and vertebrae was also affected by P status (Williams et al., 1990). Blood, bone, feces, rumen fluid and saliva can all be used with various degrees of success to indicate P status of a ruminant and reflected dietary P levels (Williams et al., 1991a,c). Aluminum and Phosphorus Interactions Aluminum is the third most abundant element in the earths crust, following silicon and oxygen, and is the most common metal found in the earths crust (OConnor et al., 2002). Aluminum is highly reactive and does not normally appear in its elemental form; instead, Al binds to other elements or compounds (McDowell, 2003). Soil Al concentrations can range from 1 to 30% but are typically in the 0.5 to 10% range by weight (OConnor et al., 2002). It is not uncommon to find high amounts of Al complexes in tropical sandy soils, binding soil P and making it unavailable for plant uptake (McDowell, 2003). Aluminum chloride (AlCl3) was added to fields of manure covered soil and reduced P runoff by 53% (Smith et al., 2004). Data such as this prove that Al can bind P and increase a soils P sorption capability. In almost all cases, Al is considered to be a toxic mineral, and is not considered to be a required element, except possibly in female rodents (McDowell, 2003). Rosa et al. (1982) reported that increases in dietary P in sheep increased feed intake while it was decreased by increases in Al and Fe. Increased dietary levels of Fe and Al in sheep diets resulted in weight losses; ADG was decreased from 156 to 97g/d in high Fe diets and from 159 to 95g/d in high Al diets. When additional P was added to diets containing high Fe or Al, ADG losses were minimized. The rationale for this response was that the diet

PAGE 21

11 being fed was borderline to deficient in P either at 0.17% to 0.23 % (NRC, 1985). Additionally, plasma P levels increased with Fe and decreased with Al diets (Rosa et al., 1982). Aluminum is not added to animal diets and, in most cases, is found in feeds only because of contamination; whereas P is often added to animal feeds and mineral mixtures. When Al is absorbed via lungs, skin and intestines, only small amounts are actually retained and can be reduced further with fluorine (F) consumption. Most Al is excreted in the feces and urine (McDowell, 2003). In a study at the University of Florida, lambs were given 2,000 ppm of an available source of Al (AlCl3) and Al tissues levels were only mildly elevated (Valdivia et al., 1982). In a similar study, calves were given 1,200 ppm of AlCl3, and performance was not influenced and changes in tissue constituents were only mildly elevated (Valdivia et al., 1978). The kidneys, liver, skeleton and brain are often the tissues affected by Al toxicities (McDowell, 2003). High dietary available Al can result in unabsorbable complexes with P in the intestinal tract. The first effect from a low dietary P level is a decline in plasma P (Williams et al., 1991a,c) further characteristics, including bone demineralization, then follow (McDowell, 2003). When dietary Al exceeds the maximum tolerable level suggested by the NRC (1985) of 1,000 ppm, animals develop characteristics of Al toxicosis. Phosphorus is the mineral primarily affected when toxic levels of Al are administered. An insoluble complex of Al and P is formed in the digestive system of the animal, binding P and making it unavailable, as seen in sheep fed high levels of Al, and signs of P deficiency resulted (Valdivia, 1977). Bone ash and bone Mg level were reduced when 1,450 ppm Al (chloride form) was given to wether lambs (Rosa et al.,

PAGE 22

12 1982). Plasma P levels in sheep given 0.15% P with no added Al were 6.9 mg/100 ml compared to 3.6mg/100ml for those receiving 2,000 ppm Al. Lambs fed 2,000 ppm Al also had lower gains and feed intakes. All animals apparent P absorption was negatively impacted except those fed high P with low Al concentrations. Correspondingly, plasma Ca levels were reduced 0.24 mg/100 ml when 2,000 ppm Al was added. Non-ruminant species are less tolerant to Al toxicity than ruminants. If the same studies were conducted on monogastric animals, toxicosis would develop using 2,000 ppm Al and would ultimately lead to death. The rational is that within the rumen Al complexes with organic anions, not affecting P radicals in the same manner as a monogastric animal (Valdivia et al., 1982). High Al concentrations have also been linked to the possible onset of Alzheimers disease. No factual evidence has been documented to prove if an Al concentration in the brain actually does affect the diseases occurrence. Through the influence of the disease in the medical field is elastic and is currently being methodically investigated (McDowell, 2003). Toxicity is primarily linked to a high Al bioavailability (McDowell, 1997; 2003; OConnor, 2002). When Al toxicity is observed in the ruminant, bone density is often impaired (Valdivia, 1977). Abnormally high amounts of bioavailable Al can also impact the status of Fe, Zn, and Mg in sheep. Dietary amounts of AlCl3 at 1,000 ppm decreased bone and kidney concentrations of Mg, additional antagonistic affects developed for P and Ca as well. (Rosa et al., 1982). Bone ash was reported by Valdivia et al. (1978) to contain lower amounts of Mg for animals fed diets containing AlCl3.

PAGE 23

13 The binding of P to WTR brings about concerns that plants will be limited in required minerals such as P, Ca and Mg because they will become unavailable. Crop yields could be negatively impacted if too much P is bound to Al or if increases in heavy metal contents are realized within the soil (Novak and Watts, 2004). If P becomes unavailable for plant uptake, deficiencies in both plants and animals could occur. Rosa et al. (1982) concluded that excessive bioavailable dietary Al increases P requirements. This may be particularly true when animals are grazing on acidic tropical pastures. In acid soils, Al and Fe become more available and both complex with P and render it unavailable to plants. Acid soils with a pH of 5 or less usually contain higher amounts of available Al and Fe (USEPA, 2003). Water treatment residuals have a pH above 5, which varies somewhat from slightly acidic to moderately basic, and alkaline sources could act as buffers to the soil. Past research has concluded that an elevated soil pH can be maintained with long term use of some WTR, that have a low Al solubility. It is unknown, but has been suggested, that WTR could have an opposite effect on the living system and could lower the pH in the digestive systems of animals which consume it directly (OConnor et al., 2002). It is well known that, in general, soils with an alkaline pH have higher mineral concentrations, than acidic soils. However, Fe, Co, Cu, Mn, and Zn are much more available in acidic compared to alkaline soils (McDowell, 1997). Many tropical soils are acidic (< 5.0 pH) with low P concentrations in forages. Acidic conditions often result in high concentrations of Al and Fe which bind other minerals (McDowell, 2003). In a study used to determine the affects of sand and soil ingestion in

PAGE 24

14 sheep, tropical soils from Costa Rica with a pH of 5.2, were shown to negatively affect the animals more than soils with higher pHs (Ammerman et al., 1984). Pollution and Phosphorus Application to Land Both the absolute number and percentages of the U.S. population employed strictly in farming has fallen dramatically over time. The pressure to produce enough food, with a smaller number of farmers, has had a worldwide impact on agricultural practices, including the expansion of agricultural into marginal lands and the over use of land in general. The agricultural industry needs to remain steadfast in providing adequate food supplies, but we must not compromise environmental, socio-economic, human, and wildlife health issues. In our effort to increase food production, pollution of our water systems has become an issue of pressing attention. In many farming practices, manure application to the land has become environmentally problematic. The majority of P applied to the land as manure often is converted into an insoluble form in the surface horizon of the soil. The accumulated P is subject to erosion or runoff following heavy rains and transported to surface water. Thus, regulations on manure application rates have developed to avoid P pollution of surface waters. (Dayton and Basta, 2001; OConnor et al., 2002; Dayton et al., 2003). Animal producers oppose new stricter regulations placed on manure use because the cost of compliance can be high. In Okeechobee County, Florida, the state has mandated environmental improvements for certain farms. The state shared 75% of the cost to update dairy facilities utilizing 456 employees and 50 million dollars (Lanyon, 1994). There are management methods that can be applied to decrease P runoff, but many are expensive when applied to large farming operations. Using an intensive management system on an average size farm of 100 head that was feeding a high quality

PAGE 25

15 pasture, reduced concentrate feeding by 16%, and resulted in a 5% lower milk yield Yet, this system also reduced the manure application to the land, lowered feed costs, and reduced manure handling procedures so that the farm was able to increase overall annual profitability by $10,000, which is equivalent to $93/cow (Rotz et al., 2002). The same method was utilized by large farms, with 800 head, and profits were increased by $23/cow, but only with an increase in milk production (Rotz et al., 2002). It is a necessity to protect the land from erosion and the water from P pollution caused by manure land application practices, but it is a struggle between the better of two interests. A study in Pennsylvania researched several species of food animals to try to determine differences among species and P production in manure. Three soils that contained manure from ruminants, swine, and poultry were evaluated. Differences in P concentrations among species could not attributed to any pertinent factor and could be assumed to be a result of initial P variations, differences in the P distribution of the soils, or the mixing of the soils and manures. Mixing of all manure types decreased P runoff and was deemed useful in reducing P losses during heavy rains. Mixing the soil and manure promotes sorption of P materials and dilutes the P in the soil surface (Kleinman et al., 2002). Ideally, soil mixing could occur on farms, but labor and machinery costs make the process unrealistic for large scale operations. The current strategies used to reduce P runoff and leaching are soil tillage, crop residue management, cover crops, buffer strips, contour tillage, runoff water impoundment and terracing. These techniques have not be proven to achieve enough success to be used solely, or cooperatively to reduce the current environmental problem (Dayton et al., 2003).

PAGE 26

16 Regulations Scrutiny from the general public and governmental agencies has developed with the increasing pollutants detected in water bodies throughout the United States. In 1995, a manure spill of 144 million liters, twice the size of the Exxon Valdez oil spill, occurred in North Carolina (USEPA, 1997). Farms in the United States are being forced to adhere to strict laws designed to protect the general public involving issues of odor control, water and food safety (Powers, 2003; Federal Registar, 2004). In 1969, Congress passed the National Environmental Policy Act (NEPA). The NEPA has two major divisions; the Council of Environmental Quality (CEQ) and the Environmental Impact Agency (EIA). The CEQ consists of a board of three members who advise the president on environmental issues. The EIA oversees legislation proposed for federal action on environmental issues (Mann and Roberts, 2000). Environmental law is governed by statutory laws and is regulated by federal, state and local administrative agencies. The Environmental Protection Agency (EPA) is the federal agency that oversees such issues, (Mann and Roberts, 2000), having jurisdiction with 10 regional offices nation wide (Meyer, 2000). According to environmental research, the sheer amount of waste generated by large animal facilities poses risk to ground and surface water (Lorentzen, 2004). According to the EPA, farming creates 455 million metric tons of manure each year (Lorentzen, 2004). In 1972, Congress amended the Federal Water Pollution Control Act (FWPCA) of 1948 with the Clean Water Act (CWA) of 1972 (Powers, 2003; Lorentzen, 2004). Again in 1977, 1981, 1987, and 2002, the CWA was amended to ensure clean water for the following: recreational use, protection of the wildlife, and to eliminate pollutants into the ground and drinking water. Concerns that embody the agricultural industry involve

PAGE 27

17 leaching and runoff of nitrogen, solids, and P into the ground water, water ways and water beds (Lorentzen, 2004). Violators of the CWA are subject to both civil and criminal charges. Criminal charges only apply if the violation was intentional. If charged criminally, the fines can range from $2,500 to 1,000,000 dollars and from one to 15 years in prison. Civil charges pertain to all other violations. Ignorance does not preclude one from dismissal of civil or criminal charges. Civil fines can reach a limit of $10,000 a day and an overall maximum of $25,000 per violation (Miller, 2004). Watersheds do not always use filtration techniques when purifying natural water sources (Rotz et al., 2002). A prime example is the New York State watershed located in the Catskill Mountains. This particular region of the state is primarily covered with forests and dairy farms, and supplies 4.5 billion liters of water to people in New York City each day (NRC, 2000). The New York watershed which provides 90% of the drinking water to the city, is purified only chemically, and serious harm could result if manure solids were to contaminate the water systems (Rotz et al., 2002). As defined by the CWA, there are two sources of pollution, point and non-point sources. Point source means there is one defined place or confined area in which the pollution has been released. Point source regulations mandate effluent limitations, based on technological advancements, on the amount of pollution which can be discharged from one source into a body of water. Concentrated animal feeding operations (CAFO s) are often considered point source pollution candidates (Meyer, 2000), and are defined as operating with 700 cattle or a total of 1,000 animals (Lanyon, 1994). Non-point source pollution occurs when the source of pollution can not be traced to a single area. Non

PAGE 28

18 point source is more often the cause of agricultural pollution; in regards to land use, run off, and leaching (Mann and Roberts, 2000). Non-point pollution may not even be observed in the watersheds that is directly affected, but may be carried for many meters down stream and damage areas with no direct contact with the original pollutant (Lanyon, 1994). Classically, farms have been identified as non-point sources of pollution. It has been predicted that within the near future, with increasing regulations and, because of public agendas and concerns, smaller farms will too, be included in point source pollution policies (Lanyon, 1994). For any type of discharge into open water ways, permits by the National Pollutant Discharge Elimination System (NPDES) are required each time and stricter rules are in the near future (Meyer, 2000). Machine and equipment regulations governing businesses involving environmental law are determined by the notion of best available control technology (BACT). This requires that procedures and machines in use need to meet EPA standards for pollution-control. New businesses need to follow standards more strictly than businesses already in existence. As technology advances, new techniques develop that make it possible to reduce pollution. New companies are legally bound to effectively alleviating pollution with the use of advanced technologies. Timetables for existing companies have been applied, meaning that the replacement of old equipment is to be implemented within a reasonable time period. The replacement equipment protocol for existing companies should then be based upon the best practical control technology (BPCT) law by replacing, rather than repairing, equipment, to meet the most current EPA standards (Miller, 2004). Many of the new regulations imposed on businesses regarding environmental safety are locally mandated by state and county polices. In Maryland, Virginia, and

PAGE 29

19 Delaware, stricter polices are being implemented in regards to P application to the land. In Maryland, all P application must abide by the Water Quality Act of 1998, which dictate soil testing to determine if the soil is saturated with P, and if manure application can be permitted. In Virginia, food animal practices are closely scrutinized, particularly the poultry industry; in Delaware manure can usually be treated once every three years to comply with soil P limitations (Penn and Sims, 2002). Water Quality Control Boards are now being mandated to more strictly adhere to the monitoring of N and P levels in the soils. In California, there is an overabundance in the pollutant count in several bodies of water of both P and N. Leaching and run off from manure enriched fertilizers is thought to be the primary cause. The reality is that this type of fertilization is a matter of convenience, availability, and cost profitability rather than providing the optimal nutrients for the flora or concern for the ecosystem (Farm Press, 2004). WTR and Environmental Uses Currently, there is no solution for the distribution of the large quantity of manure produced in the livestock industry. The major issue at hand is the confinement of large operations to small areas of land. Conflicts arrive in application and concentrations of allowable feces. Both N and P are constituents of animal waste products, and are harmful pollutants, yet federal, state, and county standards differ in the applicable uses and concentrations of manure for land, resulting in confusion as to how manure should be properly applied (Lanyon, 1994). Water treatment residuals (WTR) are by-products from water purification procedures. They are rich in metals like Al and Fe, though the exact composition can vary. The elemental levels of Al, Fe, and Ca vary when comparing WTR depending on

PAGE 30

20 the chemical used during the water treatment process and the age (or dryness) of the WTR. In turn, these differences will reflect different abilities to adsorb P (Dayton et al., 2003; Ippolito et al., 2003). During the water treatment process, a chemical, called a coagulate, is added to the water and later forms WTR. This addition of chemicals to water will cause a reaction and form a flocculent precipitate, which coats small particles, such as clays making them more likely to be removed by sedimentation or filtration. Aluminum sulfate (iron sulfate, or calcium sulfate) coagulates may be added to raw water, (the WTR of interest for all further discussion is Al based). The water is then circulated with vigor to uniformly disperse the Al product. Aluminum reacts readily with alkaline products within the water and produces an Al hydroxide solid, which has entrapped impurities. The sedimentation process allows the solids to settle-out. These solid by-products are Al oxides bound to clay size particles and are known as WTR. The processes of coagulation and sedimentation usually precedes filtration in a water treatment plant, and serves to reduce turbidity and increase the efficiency of bacterial removal by filtration (Dayton and Basta, 2001; Brady and Weil, 2002; Ippolito et al., 2003; Water Resources, 2005). The physical characteristics of these WTR are similar to top soils (Haustein et al., 2000). The use of WTR and metal-binding by-products could be one solution to the accumulation of soluble P in the top layer of soil, which leads to nonpoint pollution during heavy rains (Penn and Sims, 2002). In particular, Al containing WTR would benefit sandy soils low in organic material. Sandy soils tend to provide little P retention capabilities and runoff is likely (Penn and Sims, 2002). Soils that are saturated with P may also benefit from WTR application. It has been shown that P saturated soils are

PAGE 31

21 unable to hold added P and thus will result in P ground water complications (Penn and Sims, 2002). Added Al in the form of WTR may help depress P runoff by increasing soil P retention capabilities (OConnor et al., 2002; Penn and Sims, 2002). Publications in 2002 indicated a reduction in P leaching with the addition of Fe and Al from biosolids, claiming that metal oxides formed lead to increased P retention (Soon and Bates, 2002). Research at the University of Florida concluded that increases in dietary Al levels reduced feed intake, gains and P plasma concentrations in sheep. The Al given to these animals was in the form of AlCl3. The impact of additional Al was not positive for animal gains as ADG was 105 and 148 g/d for those consuming a high Al or a low Al diet, respectively. When additional dietary P was given, the ADG increased, but it was not as high as for animals not consuming any Al (Rosa et al., 1982). These results demonstrate the capabilities Al had to lower P status in the animal, but it is unknown what will occur if a less bioavailable form of Al is fed. Other mineral plasma concentrations were also impaired with increased dietary Al. Magnesium content was depressed in the kidneys, and bone of those animals receiving the high dietary Al (Rosa et al., 1982). Similar results using Mg have also been documented at Rutgers University in avian species. Young chicks and mallard ducks when fed high Al diets, as AlCl3, had a high incidence of P binding, lowered P serum levels, depressed growth, lowered tibia weights and lower bone mineralization (Capdevielle et al., 1997). Few studies have been conducted to determine the results of P accumulation, ground water pollution, and the quantity of Al which is capable of binding P in WTR when consumed by ruminants. The majority of studies in regards to WTR and Al content have been focused on the ecological risks associated with plants in acidic soils

PAGE 32

22 (OConnor et al., 2002). Studies involving soil P binding mechanisms have also proven to be helpful. Phosphorus absorption capacity was increased by 20 times with the use of WTR when compared to high Al clay (Haustein et al., 2000). Applications involving pollution control with the use of WTR fed to sheep will be implemented here to compare the bioavailability of Al from WTR to an available source of Al (AlCl3) and evaluate how Al affects the performance of growing sheep.

PAGE 33

CHAPTER 3 EFFECT OF ALUMINUM-WATER TREATMENT RESIDUALS ON PERFORMANCE AND MINERAL STATUS OF FEEDER LAMBS Introduction Ingestion of highly available dietary Al (e.g. AlCl3) by livestock may result in P deficiency. Aluminum toxicity is often observed as a P deficiency (Valdivia, 1977). Additionally, high amounts of bioavailable Al can also impact the status of Fe, Zn, and Mg in sheep (Rosa et al., 1982). Under grazing conditions, ruminants typically consume 10% to 15% of their DM intake as soil (Field and Purves 1964; Healy, 1967; 1968). In sheep dietary Al suppressed voluntary feed intake, feed efficiency, plasma P, growth, and gains (Rosa et al., 1982). When additional P was ingested, these negative effects were less severe but were still evident. Water treatment residuals (WTR) are the byproducts from a water purification procedure, and can contain high amounts of Al, Fe or Ca; here they contain high amounts of Al and has a high P sorption capacity. The bioavailability of Al in WTR varies, but is generally low and thought to be harmless (OConnor et al., 2002). Since Al is highly reactive and has been shown to chemically bind P, the administration of WTR on manure containing soils could be a solution for P pollution of water systems by increasing soil P retention capabilities (Penn and Sims, 2002). Concerns occur because of possible ingestion of the WTR by grazing animals and the reaction of Al and P in a low pH 23

PAGE 34

24 system. No previous research has been conducted to determine the potential toxicity of WTR when directly consumed by grazing ruminants. The purpose of this study was to determine if feeding growing lambs a bioavailable source of Al (AlCl3) versus a less available source of Al from a WTR would affect growth, feed intake, plasma P levels, tissue concentrations, and apparent P absorption. Materials and Methods Animals, Diets, and Management Forty-two, wether (30) and female (12), five to eight-mo-old lambs, (22 Suffolk and 14 Suffolk-crosses) were utilized in a 111-d experiment at the University of Florida Sheep Nutritional Unit located in Gainesville, Florida. The experiment was conducted from June 6th until September 25, 2004. The lambs weighed between 22 to 39 kg at d zero. Lambs were shorn on d 42 in an attempt to combat heat stress and to increase optimum feed intake. Prior to the experiment, lambs were vaccinated with an 8-way Clostridial given as an injection of 2-mL, four wks apart (Ultra Choice 8; Pfizer Animal Health, Exton, PA) and were dewormed, with two 1 mL doses of Ivermectin, two wks apart (Ivomec; Merial Ltd., Iselin, NJ). To prevent coccidiosis, an amprolium solution was given as an oral drench, lambs received 1 mL daily in a six d sequence (Corid 9.6%; Merial, Duluth, GA). On d 21 the animals were dewormed orally with 5cc of Fenbendazole, (Panacur; Pfizer Animal Health, Exton, PA) and again drenched with 1 mL of Corid from d 21 to 26 (Corid 9.6%). The lambs were housed (seven to each pen), in covered, earth-floored wooden pens (24 sq. m), bedded with pine wood chips with adequate bunk space and ad libitum water and common salt. The University of Florida Institutional Animal Care and Use committee approved the experimental protocol (D231) used in this study.

PAGE 35

25 A corn-SBM basal diet was formulated to meet NRC (1985) requirements for CP, TDN, vitamin, and minerals for lambs of this weight and age (Table 3-1). Prior to the experiment, during a three wk adjustment period, lambs were fed the basal diet at 1200 to 1300 g/d per animal. During the experiment, the animals were fed once daily, 1300 to1600 glamb-1d-1. Lambs were stratified by sex and randomly assigned to six dietary treatments; 1) control (10% sand), 2) (9.7% sand and 0.3% AlCl3), 3) (2.5% WTR and 7.5% sand), 4) (5% WTR and 5% sand), 5) (10% WTR and 0% sand), and 6) (10% WTR, 0% sand, plus double the quantities of the mineral-vitamin premix, and 1.29% dicalcium phosphate). The WTR used contained 7.8% total Al on a DM basis. Ten percent of each diet was either sand, WTR, AlCl3 or a combination of the three. The diet concentrations of Al were 910, 2000, 2000, 4000, 8000, and 8000 ppm, (DM basis) respectively, for the six diets. On d 91, animals were placed into individual metabolic crates (1.4m2) to determine apparent digestibility of P. During a subsequent 21 d crate confinement, all animals were individually fed their respective experimental diets. Fresh feed was given ad libitum each morning. Orts were weighed back daily. Individual feed intake, ADG, and BW differences from wk 11 to wk 14, were evaluated. Sample Collection, Preparation, and Analyses Blood samples (jugular venipuncture) and lamb weights were collected on d 0 and every 14 d thereafter. Blood was collected (10mL) with a 20 x 1 vacutainer (Vacutainer; Becton Dickinson, Franklin Lakes, NJ) needle into evacuated tubes containing sodium heparin. Immediately after collection, blood was centrifuged at 700 x g for 30 min, and plasma was collected and frozen at 0 C. After a 30 min thaw period, to allow plasma to

PAGE 36

26 reach ambient temperature the proteins were separated using 10% trichoroacetic acid (Miles et al, 2001). On d 91, wether lambs were fitted with cloth fecal collection devices for the study of apparent digestibility of P. Feces were collected daily for 14 d and composite samples were frozen at 0 C. Each composite sample was sub-sampled and ground in a blender with stainless steel blades. Feces were then dried for 16 h at 105C to determine DM. Samples were then ashed in a muffle furnace at 600C for 8 h, digested in HCl, filtered, and diluted for colorimetric P determination (Harris and Popat, 1954). On d 111, all animals were sacrificed at a USDA approved facility. The following tissues were collected and analyzed for Al, Ca, Cu, Fe, Mg, Mn, P, and Zn contents: blood plasma, liver, heart, kidney, and brain and Se was analyzed for the kidney. Samples were dried, weighed, ashed, and solubilized in HNO3 acid (Miles et al., 2001). Bone was analyzed for P, Ca, and Mg. For all samples, P was analyzed using a colorimetric procedure (Harris and Popat, 1954). Kidney Se was determined using fluorometric procedures (Whetter and Ullrey, 1978). Calcium, Fe, Mg, Cu, Mn, and Zn in tissues and feed samples were analyzed by flame atomic absorption spectrophotometry (Perkin-Elmer Model 5000, Perkin-Elmer Corp., Norwalk, CT). Aluminum concentrations were analyzed in diets, heart, brain, liver, and kidney by atomic absorption spectrophotometer using nitrous oxide-acetylene flame (Varian SpectrAA 220 FS; Varian Inc., Walnut Creek, CA). Statistical Analysis Soft tissue, fecal, and feed intake data were analyzed for treatment effects using PROC GLM in SAS (SAS for Windows v9; SAS Inst., Inc. Cary, NC) in a completely randomized design. PROC MIXED of SAS was used to analyze treatment effects on

PAGE 37

27 BW, ADG, and plasma P as repeated measures with a variance component covariance structure in respect to d and subplot of animal nested within treatment. Significance was declared at P < 0.05 and tendencies were discussed when P < 0.15. Results Six animals died during the experiment. The cause of death was determined to be parasite infestation of the gastrointestinal tract, and was deemed unrelated to dietary treatment. Body weights increased for all treatments for wks 0 to 14 (Table 3-2). Average daily gains (Table A-1) and feed intakes (Table 3-3) also increased with time (P < 0.05). Throughout the experiment, lambs fed 2,000 ppm Al via AlCl3 consistently had numerically lower BW than all other treatments. During wk 6, lambs fed 2,000 ppm Al via AlCl3 had lower BW than control animals and, lambs fed 2,000 ppm Al, 4,000 ppm Al or 8,000 ppm Al from WTR (P < 0.05). Lambs receiving 2,000 ppm, 4,000 ppm and 8,000 ppm Al via WTR were heavier than animals consuming 2,000 ppm Al via AlCl3 during wk 11 (P < 0.05). Body weights during wk 11 differed by 11.3 kg, (P < 0.05) between those animals consuming 2,000 ppm Al via AlCl3 and those fed 8,000 ppm Al via WTR, whereas the difference between these two groups at wk 14 was 7.4 kg (P= 0.008). During wk 2, ADG of lambs given 8,000 ppm Al from WTR exceeded animals given 2,000 ppm Al via AlCl3 (P < 0.05). Lambs receiving 4,000 ppm Al from WTR tended (P = 0.11) to gain more than lambs fed 2,000 ppm Al via AlCl3. During wk 4 lambs receiving the control, 2,000 ppm Al via WTR and 8,000 ppm Al via WTR treatments had higher gains than lambs in the treatment given 2,000 ppm Al via AlCl3 (P < 0.05). During wk 6, lambs consuming the control, and 4,000 ppm Al from WTR diets gained more than lambs consuming 2,000 ppm Al from AlCl3 (P < 0.05). Additionally,

PAGE 38

28 during wk 6, all treatments, except 2,000 ppm and 4,000 ppm Al via WTR had gains much lower than the control (P < 0.05). During wk 11, animals began a 3-wk individual feeding regime to determine feed intake. From wk 11 to wk 14, lambs fed the control, 2,000 ppm, and 4,000 ppm Al via WTR (P < 0.05) consumed more than those fed 8,000 ppm Al via WTR. Observations of plasma P during wk 4 (Table 3-4) showed that animals receiving 2,000 ppm Al via WTR had higher concentrations than all other treatments, except those receiving 8,000 ppm Al via WTR plus double the minerals and vitamins (P < 0.05). The animals receiving 8,000 ppm Al via WTR had lowest plasma P of all groups of animals (P < 0.05). In wks 6 to 11, the lambs receiving 2,000 ppm Al via AlCl3 had lower plasma P than controls (P < 0.05). During wk 11, both the control and lambs receiving 2,000 ppm Al via WTR had higher plasma P than animals receiving 2,000 ppm Al via AlCl3 (P < 0.05). Analyses of plasma P during wk 14 showed that controls had higher P concentrations than lambs receiving 4,000 ppm Al via WTR, 8,000 ppm Al via WTR or 8,000 ppm Al via WTR plus two times the amount of added mineral-vitamin premix, and 1.29% dicalcium phosphate (P < 0.05). Plasma evaluations of all other minerals showed, no differences among treatments, which included the following (g/ml): Ca 87 to 101, Mg 17 to 21, Cu 1.3 to 1.5, Fe 0.9 to 2.0, Mn 0.05 to 0.06, and Zn 0.3 to 1.6. Tissue mineral concentrations (Table 3-5) among treatments were deemed not to be hazardous to animal health. With the exception of Cu, tissue mineral concentrations remained within normal ranges (Miles et al., 2001). Liver Cu concentrations were high for all treatments. The mineral-vitamin premix used, inadvertently contained excess Cu in relation to sheep requirements. Levels of P showed no differences among treatments

PAGE 39

29 except that animals given 4,000 ppm Al from WTR deposited more P in the kidney than those animals receiving 8,000 ppm Al from WTR (P < 0.05). No differences (P < 0.05) were observed in soft tissue or bone Ca concentrations. Aluminum was deposited in lower amounts in the brain for lambs fed 2,000 ppm Al via WTR than all other treatments except the control (P < 0.05). Kidney Al deposits were higher in lambs receiving 2,000 ppm Al via AlCl3 than those receiving 8,000 ppm Al via WTR (P < 0.05), and those receiving 8,000 ppm Al via WTR plus two times the added amount of mineral-vitamin premix, and 1.29% dicalcium phosphate (P < 0.05). Concentrations of Mg showed no differences in soft tissue deposition. Differences in Fe deposition were observed in liver (P < 0.05), with lambs consuming the AlCl3 treatment having lower Fe concentrations than those receiving the two treatments of 8,000 ppm Al as WTR. Variations in heart and kidney Mn concentrations seemed unrelated to Al source or quantity. Apparent P absorption ranged from -12.9 to 31.8 % (Figure 3-1). The control and all WTR treatment lambs had a greater apparent P absorption (10.9-31.89%) than the negative absorption (-12.9%) of lambs fed 2,000 ppm Al via AlCl3 (P < 0.001). Discussion Increases in BW, ADG and intakes were observed for all treatments and can be likely attributed to increased appetite which occurs in growing animals. The previous studies at the University of Florida conducted by Valdivia et al. (1978; 1982) observed an increase in feed intake from 1.03 to 1.20 g/d, and an increase in BW gain as dietary P was increased from 0.15 to 0.29 % in diets that contained 1,200 ppm to 2,000 ppm Al as AlCl3. Valdivia et al. (1978) and Rosa et al. (1982) concluded that the increase in P was able to overcome the clinical signs normally observed with Al toxicosis. Diets in the present study contained approximately 0.25% P as fed (Table 3-1), which exceeds the

PAGE 40

30 requirements (0.23% dietary P) of lambs of this age and breed (NRC, 1985; McDowell, 2003). Our study showed no major losses in weight or intakes regardless of treatment, which seems to be attributed to the proper amounts of dietary P (0.25%) supplied. This concurs with the work of Valdivia et al. (1978) and Rosa et al. (1982). The control lambs, which received 910 ppm Al from sand, and lambs receiving treatments containing WTR had no declines in intake. This is likely attributed to the low bioavailability of Al in WTR and sand (OConnor et al., 2002; Dayton et al., 2003). A low bioavailable Al source is much less likely to depress intake because the Al would not readily react with the P in the gastrointestinal tract. Aluminum from AlCl3 is an available source and has been shown to depress intakes. Declines in intakes caused by ingestions of an available Al source have been observed in various species including: sheep (Valdivia et al., 1978; Rosa et al., 1982), broilers and chicks (Fethiere et al., 1990), humans (Chappard et al., 2003; Rengel, 2004) and rats (GmezAlonso et al., 1996). An Al toxicity results in a P deficiency (McDowell, 2003) which can lead to serious tissue damage, lower intakes and gains. Williams et al. (1990; 1991a,c; 1992) induced a P deficiency in heifers and observed an 11% decrease in feed intake. In the present study, there was a decrease in feed intake for the lambs that were fed 2,000 ppm Al via AlCl3. This is expected, as AlCl3 is considered to be a bioavailable source of Al (Valdivia et al., 1978; Rosa et al., 1982), and thus may induce a P deficiency and depress feed intake. Ingestion of Al as AlCl3 by ruminants decreases bone density, plasma P levels, feed intakes and gains (Rosa et al., 1982; Valdivia et al., 1982; Ammerman et al., 1984). Animals receiving the AlCl3 diet repeatedly had lower BW and feed intakes than animals

PAGE 41

31 fed other sources of Al. Lower intakes and gains can be attributed to Al availability, similar observations occurred when 0.75% aluminosilcate was fed to laying hens and feed intake was significantly depressed (Fethiere et al., 1990). One of the objectives of the present study was to compare the availability of Al in WTR to Al in AlCl3 and a control when fed to ruminants. During wk 11, body weights ranged from 36.8 kg for lambs fed 2,000 ppm Al via AlCl3 diet to 48.1 kg for lambs fed 8,000 ppm Al via WTR. Thus, lambs receiving 8,000 ppm Al from WTR, on average, had BW that were 11.2 kg heavier than those fed 2,000 ppm Al from AlCl3 despite the four fold difference in total Al administered. The group fed 8,000 ppm Al from WTR had the highest amount of Al and the largest percentage of WTR (10% of the diet as fed). Differences observed in BW, between lambs fed 8,000 ppm Al via WTR and 2,000 ppm Al via AlCl3 validates previous studies which showed Al in Al-WTR to be high in a non-available source of Al (OConnor et al., 2002; Novak and Watts, 2004) and that AlCl3 is available for uptake in the small intestine (Valdiva et al., 1978; Rosa et al., 1982). It is thought that grazing ruminants can consume up to 10-15% of their total DM intake as soil (Healy 1967; 1968). It has also been shown that soil Al is often consumed by grazing ruminants in amounts as high as 10% of the soil consumed. Aluminum ingested from soil sources has not been shown to reduce performance. Ammerman et al. (1984) fed sheep varying soils types, from Latin America, containing as much as 16,600 ppm Al. They concluded that the soil Al sources had no significant effect on BW, gains, and intakes of the sheep which consumed them. The soils contained various levels of Al or Fe oxides, which is similar to the chemical form of Al from WTR. The additions of high Fe and Al soils had no harmful effects on P utilization, feed intake, or gains.

PAGE 42

32 Differences, in general, between treatments were limited throughout the trial. Lambs receiving diets containing Al via WTR at varying levels showed no differences in BW from the control (P < 0.05). Additions of WTR in amounts as high as 10% of the diet, and representing 8,000 ppm Al in the diet, do not negatively impact growing lambs in relation to BW, ADG, and feed intakes when dietary P is at least 0.25%. Thus, under natural grazing conditions, [where 10% of the DM intake is of soil (Field and Purves, 1964; Healy 1967; 1968)], even high rates of surface applied WTR are not expected to harm animal performance. During wk 14, the ADG of treatments plateaued, consistent with a natural sigmoidal growth curve. Prior to wk 14, animals were gaining at rates between 463 to 593 g per d. The rate declined during wk 14 to only 207 to 244 g per d, but the decline is not attributed to dietary treatments. Animals appeared healthy with notable accumulations of body fat. Lambs in both the control and AlCl3 treatment continued to gain larger amounts of weight during wk 14, because they had not reached a maintenance weight. Lambs fed 2,000 ppm Al via AlCl3 had lowered growth, intake and BW throughout the trial and had not reached a growth plateau by wk 14. The control animals during wk 6 experienced an illness which was attributed to parasite infestations which suppressed ADG means thereafter. In previous studies, similar declines in ADG were observed with AlCl3 additions, and animal growth peaked at later dates than those not receiving an Al source (Valdivia et al., 1978; Rosa et al., 1982; Fethiere et al., 1990). Intakes, regardless of treatment, increased with time. Constituents added to the basal diets did not cause any animals to become anorexic, a common clinical sign of Al toxicity, or P deficiency (Williams et al., 1992; McDowell, 2003). Differences in intakes

PAGE 43

33 were evaluated individually in a 3-wk period between wk 11 to 14. Prior to this date, lambs had been group fed. Individual intake data were similar to those reported by Rosa et al. (1982), and Valdivia et al. (1978). Lambs fed diets containing 2,000 ppm Al from AlCl3 consumed less than the control (P > 0.05), which can again be attributed to the high bioavailability of AlCl3. Intakes were the lowest for animals consuming 8,000 ppm Al from WTR. During wk 14, these animals had the highest BW, but a decline in ADG from wk 11 (480 g) to wk 14 (207.0 g), which was the lowest gain for that period. Intakes for lambs receiving 8,000 ppm Al from WTR were lower than the control, 2,000 ppm and 4,000 ppm Al from WTR (P < 0.05), but higher than lambs receiving 2,000 ppm from AlCl3 or 8,000 ppm Al from WTR with additional minerals and vitamins, (P > 0.05). Prior to wk 14, lambs fed 8,000 ppm Al from WTR showed adequate performance in relation to gains, intake and BW. Therefore, the cause of these declines seen in lambs receiving 8,000 ppm Al via WTR are unknown and could be related to normal growing patterns, an unknown parasite infestation, Cu toxicities, Al toxicities, or other various environmental interactions. During wk 4, lambs receiving 2,000 Al from WTR had the highest concentration of plasma P and differed from the control, those receiving 2,000 ppm Al from AlCl3, 8,000 ppm Al from WTR. (P < 0.05). Huff et al. (1996) administered 3.7% aluminum sulfide to broiler chicks and observed a declines in serum P after a 3 wk period. Lambs in the present study, had plasma P levels decline from 54.2 g/ml to 19.6 g/ml, between wk 4 and wk 8. Additionally, all treatments showed declines in plasma P during this period, but the AlCl3 treatment declines were most often the greatest. During wk 11 and 14, plasma P concentrations began to increase in all treatments. One could conclude that

PAGE 44

34 plasma P concentrations declined to levels which demanded the use of body stores of P (Williams et al., 1990; McDowell, 2003). Bone mineral content was evaluated in the long bones, with no differences among treatments and no evidence of a mineral depression; yet research has shown that the ribs and the vertebrae are first to become depleted in mineral concentrations (Williams et al., 1990; 1991a; McDowell, 2003). Therefore the possibility exists that increases in plasma P levels during wk 11 to 14 occurred from bone mineral resorption. This is unlikely, but not unreasonable, because within a long time frame of 8 wk (between wk 6 and wk 14) bone loss most likely would have been observed in the long bone of the leg which was analyzed. Previous experimental data have not demonstrated similar results by showing an increase in plasma P after a decline. Therefore, observations are speculative at this time and further research is needed to validate this theory. Tissue mineral concentrations analyzed for this study were in the normal ranges for lambs of this breed and age (Miles et al., 2001; McDowell 2003). Previous research found differences in kidney, bone, liver and spleen concentrations of Al, Fe, P Mg and Zn (Rosa et al., 1982) and Ca (Rosa et al., 1982; Zafar et al., 2004) when various amounts of Al were fed. In the present study, Al concentrations differed in brain, heart, liver, and kidney, Mg in bone, and Fe in the liver. Absorption of Al in mongastrics is approximately 0.1% (Rengel, 2004) and is thought to be even lower in ruminants (Valdivia et al., 1970; 1978). Aluminum accumulation occurs most readily in the brain. The exact mechanism is unknown but Al can cross the blood-brain barrier (Rengel, 2004). Accumulations of Al in brain tissue were greater from lambs fed 2,000 ppm Al from AlCl3, than from lambs fed 2,000 ppm from WTR (P < 0.05). Aluminum

PAGE 45

35 concentrations in brains increased when Al from WTR was fed at levels higher than 2,000 ppm, but did not differ from the control. Liver depositions of Al were highest in lambs fed 8,000 ppm Al from WTR (P < 0.05). In the kidney, the highest concentrations of Al were detected when lambs were fed 2,000 ppm Al from AlCl3, and differed from both treatments receiving 8,000 ppm Al from WTR and from the control and 2,000 ppm Al via WTR (P < 0.05). Rosa et al. (1982) observed increases in Al tissue concentration as Al consumption increased, which was not consistently observed in our study. Additionally, soft tissues, except brain matter, that have been evaluated in past studies have not been shown to accumulate large amounts of Al during short time periods (Rengel, 2004), and may not prove to be useful for determination of differences of any Al sources and levels. Apparent P absorption from a 14 d fecal collection showed differences among all five treatments versus the treatment containing 2,000 Al via AlCl3. Studies by Valdivia et al. (1982) observed a marked decrease in P absorption and net P retention in lambs fed 2,000 ppm Al as AlCl3. Negative apparent P absorptions were observed in all groups except those given high P with low Al. When 0.29% P was fed with no dietary Al, the mean apparent absorption was unaffected. In our study, the control had an apparent absorption of 22.5%, and the mean for all the WTR groups was 21.2%. This suggests that Al in WTR did not negatively impact or reduce dietary P absorption. Valdivia et al. (1982) found a negative apparent P absorption (-10.7%) when 0.29% dietary P and 2,000 ppm Al as AlCl3 were fed to sheep. Additionally, Martin et al. (1969) conducted P retention studies using dietary applications of a hydrated Al source and discovered that when Al was fed to sheep, retained amounts of P decreased linearly, as Al fed increased

PAGE 46

36 from 910, 2000, 4000, 8000, and 8000 ppm. Similar results were observed in our study when 2,000 ppm Al was added via AlCl3 to the basal diet which contained 0.25% P. The apparent P absorption averaged 12.9% at wk 14 and therefore suggested a negative impact on dietary P utilization with added Al as AlCl3. Summary and Conclusions A 14 wk experiment was conducted using 36 lambs. Individual feeding was recorded between wk 11 to 14. Diets, containing 0.25% P (as fed), included 1) control (10% sand), 2) (9.7% sand and 0.3% AlCl3), 3) (2.5% WTR and 7.5% sand), 4) (5% WTR and 5% sand), 5) (10% WTR and 0% sand), and 6) (10% WTR, 0% sand, double the added quantities of the mineral-vitamin premix, and 1.29% dicalcium phosphate). The Al varied from 910 to 8,000 ppm among diets. Lambs fed the control and WTR had no decline in intake, but the AlCl3 lambs repeatedly had lower BW and intakes. The WTR contain a non-available source of Al and did not cause performance declines Additions of this WTR respecting Al concentrations as high as 8,000 ppm, did not negatively impact growing ruminants in relations to BW, ADG, and intakes. During wk 6, all treatments showed declines in plasma P, but the AlCl3 treatment was often the lowest, and during wk 11 plasma P began to increase. Accumulations of Al in the brain were greatest for lambs given 2,000 ppm Al from AlCl3 and increased numerically when Al as WTR was fed at levels higher than 2,000 ppm. With the exception of the brain, soft tissues did not accumulate large amounts of Al during this 14 wk experiment. Apparent P absorption from a 14 d metabolic study was positive (10.9-31.8%) for all lambs fed the control and various levels of WTR. However, lambs that received 2,000 ppm Al via AlCl3 had a negative P absorption of -12.9 %. This was a lowered (P < 0.03)

PAGE 47

37 P absorption compared to all other treatments. Aluminum, as AlCl3, fed at 2,000 ppm reduced dietary P retention, but varying amounts of Al as WTR had no effect on P apparent absorption with similar absorption rates as the control. Therefore when dietary P is supplied in amounts of 0.25% or higher, Al (as WTR) fed to lambs in amounts as high as 8,000 ppm did not negatively impact the feed intake, gain, BW or P absorption. Implications Dietary administration of AlCl3 has negative impacts on ADG, BW, feed intake, apparent absorption of P, and P plasma concentrations. Lambs fed WTR had apparent P absorption percentages that were similar to the control and were higher than the AlCl3 treatment. Water treatment residuals are not harmful when consumed in amounts up to 8,000 ppm Al, when P is supplied in amounts of 0.25%, and do not negatively affect gain, feed intake, BW, or P availability.

PAGE 48

38 Table 3-1. Diet composition (as-fed) and analyses for average (n=18) concentrations for macroand micro-elements for treatments Treatmentsa Ingredient (%,as fed) 1 2 3 4 5 6 Ground Corn 41.1 41.1 41.1 41.1 41.1 39.9 Soybean hulls 12.5 12.5 12.5 12.5 12.5 12.5 Wet molasses, unfortified 10.0 10.0 10.0 10.0 10.0 10.0 Cottonseed hulls 8.0 8.0 8.0 8.0 8.0 8.0 Corn gluten meal, 60% CP 5.5 5.5 5.5 5.5 5.5 5.5 Alfalfa meal, 17% CP 5.0 5.0 5.0 5.0 5.0 5.0 Vegetable oil (soybean) 4.0 4.0 4.0 4.0 4.0 4.0 Sandb 10.0 9.3 7.5 5.0 Water treatment residualc 2.5 5.0 10.0 10.0 Aluminum chloride 0.7 Salt 1.0 1.0 1.0 1.0 1. 1.0 Urea 1.6 1.6 1.6 1.6 1.6 1.6 Ground limestone 0.7 0.7 0.7 0.7 0.7 0.7 Ammonium chloride 0.5 0.5 0.5 0.5 0.5 0.5 Flowers of sulfur 0.01 0.01 0.01 0.01 0.01 0.01 Mineral-Vitamin-premixd 0.01 0.01 0.01 0.01 0.01 0.02 Dicalcium phosphate 1.3 Analyses (ppme) Ca 7,170 7,120 7,220 7,440 7,300 10,000 Mg 2,780 2,730 2,700 2,880 2,870 3,020 Na 4,060 3,240 3,030 3,000 3,410 3,110 K 4,180 5,210 3,960 4,560 4,440 4,380 P 2,520 2,490 2,550 2,480 2,460 5,020 Al 910 2,320 2,270 3,970 7,860 7,790 Co 7 5 6 5 5 8 Cu 31 33 33 32 34 42 Fe 66 65 67 66 66 70 Mn 11 13 13 13 14 19 Zn 74 70 71 70 67 79 aDietary treatments were created by additions to a corn-SBM basal diet as follows: 1) (Control) 10% sand; 2) 9.3% sand + 0.7% AlCl3; 3) 7.5% sand, + 2.5% WTR; 4) 5% sand + 5%WTR; 5) 10% WTR; 6) 10% WTR + two times the added amount of mineral-vitamin premix + 1.29% dicalcium phosphate; Treatments 2 and 3 were formulated to contain 2,000 ppm Al, treatment 4 was formulated to contain 4,000 ppm Al, and treatments 5 and 6 were formulated to contain 8,000 ppm Al. b Sand contained 0.2 % Fe, 0.01 % Al, 0.09% Ca, 0.03 % Mg, 0.1 % P, 0.005 % Mn, 0.004 % Cu, and 0.001 % Zn. c Water Treatment Residuals contained 0.30 % Fe, 7.8 % Al, 0.11 % Ca, 0.024 % Mg, 0.3 % P, 0.004 % Mn, 0.73 % S, 0.006 % Cu, and 0.002 % Zn. dMineral-Vitamin-premix contained 8.0% Mg (as oxide), 0.70% Fe (as sulfate), 2.40% S (as sulfate), 1.9 % Cu (as sulfate), 6.0 % Mn (as oxide), 0.47 % I (as iodate), 0.075 % Se (as sodium selenite), 4.5 % Zn (as oxide and sulfate), 133,363.4 IU/kg Vitamin A supplement, 412,272.7 IU/kg Vitamin D3 supplement, 259.1 IU/kg Vitamin E supplement, rice mill byproduct, and stabilized fat as a vitamin carrier. eDry matter basis.

PAGE 49

39 Table 3-2. Effects of dietary Al concentration and source on BW of feeder lambsa Treatmentb 1 2 3 4 5 6 SE BW, kg Wk 0 32.6 31.4 33.1 31.1 32.2 31.7 1.5 2 32.8cd 31.6c 34.6cd 34.4cd 37.5d 33.6cd 1.9 4 34.3cd 31.6c 37.7de 35.8cde 41.3e 35.2cd 2.3 6 38.4d 32.3c 40.7d 39.3d 41.2d 34.7cd 2.6 11 41.4cd 36.8c 46.7d 45.1d 48.1d 42.9cd 2.5 14 49.3cd 45.9c 52.8d 50.3d 53.3d 49.7cd 1.9 aData represent least squares means; n = 7 per treatment bDietary treatments were created by additions to a corn-SBM basal diet as follows: 1) (Control) 10% sand; 2) 9.3% sand + 0.7% AlCl3; 3) 7.5% sand, + 2.5% WTR; 4) 5% sand + 5%WTR; 5) 10% WTR; 6) 10% WTR + two times the added amount of mineral-vitamin premix + 1.29% dicalcium phosphate; Treatments 2 and 3 were formulated to contain 2,000 ppm Al, treatment 4 was formulated to contain 4,000 ppm Al, and treatments 5 and 6 were formulated to contain 8,000 ppm Al. cdeMeans within rows lacking a common superscript differ (P < 0.05). Table 3-3. Effect of dietary Al concentration and source on feed intake of feeder lambsa Treatmentb 1 2 3 4 5 6 SE intake, glamb-1d-1 Wk 2 827 959 1170 1120 1100 1020 4 1410 876 1150 1150 1070 1120 6 954 1110 1150 1200 1210 1210 11 1610 1460 1790 1550 1910 1940 14 1940c 1870cd 1900c 1940c 1270d 1570cd 54.6 aData represent means of intake during wk 0 to 11 and least squares means during wk 14; n = 7 per treatment bDietary treatments were created by additions to a corn-SBM basal diet as follows: 1) (Control) 10% sand; 2) 9.3% sand + 0.7% AlCl3; 3) 7.5% sand, + 2.5% WTR; 4) 5% sand + 5%WTR; 5) 10% WTR; 6) 10% WTR + two times the added amount of mineral-vitamin premix + 1.29% dicalcium phosphate; Treatments 2 and 3 were formulated to contain 2,000 ppm Al, treatment 4 was formulated to contain 4,000 ppm Al, and treatments 5 and 6 were formulated to contain 8,000 ppm Al. cd Lambs were individually fed for 3 wk ending at wk14; Means within rows lacking a common superscript differ (P < 0.05).

PAGE 50

40 Table 3-4. Effect of dietary Al concentration and source on plasma P of feeder lambsa Treatmentb 1 2 3 4 5 6 SE g/ml, Wk 0 48.7 44.6 51.2 40.8 45.6 43.8 3.7 2 48.3 44.7 50.5 41.5 45.7 44.1 5.3 4 54.3d 54.2d 64.4c 49.8d 39.7e 58.5cd 4.6 6 39.2c 25.2d 27.9d 28.5d 26.2d 27.0d 2.5 8 33.8c 19.6d 19.9d 26.3cd 21.9d 28.1cd 2.3 11 36.2cd 22.2e 46.3c 30.0de 29.0de 29.3de 5.0 14 38.3c 34.0cd 34.9cd 28.0de 24.5de 24.9e 3.6 aData represent least squares means; n = 7 per treatment bDietary treatments were created by additions to a corn-SBM basal diet as follows: 1) (Control) 10% sand; 2) 9.3% sand + 0.7% AlCl3; 3) 7.5% sand, + 2.5% WTR; 4) 5% sand + 5%WTR; 5) 10% WTR; 6) 10% WTR + two times the added amount of mineral-vitamin premix + 1.29% dicalcium phosphate; Treatments 2 and 3 were formulated to contain 2,000 ppm Al, treatment 4 was formulated to contain 4,000 ppm Al, and treatments 5 and 6 were formulated to contain 8,000 ppm Al. cdeMeans within rows lacking a common superscript differ (P < 0.05).

PAGE 51

41 Table 3-5. Tissue mineral composition resulting from experimental dietsa Treatmentb 1 2 3 4 5 6 SE Macro elements, (%) Ca Heart 0.01 0.01 0.01 0.01 0.01 0.02 0.004 Kidney 0.06 0.04 0.08 0.08 0.06 0.05 0.02 Liver 0.05 0.04 0.04 0.05 0.04 0.05 0.01 Bone 39.9 39.4 38.1 39.5 37.0 37.7 18.3 Mg Heart 0.15 0.13 0.15 0.14 0.12 0.13 .019 Kidney 0.11 0.11 0.11 0.13 0.10 0.10 0.02 Liver 0.066 0.062 0.066 0.070 0.062 0.064 0.004 Bone 0.79c 0.70cd 0.79c 0.79c 0.73cd 0.61d 0.04 P Heart 1.16 1.03 1.16 1.16 1.09 1.01 0.14 Kidney 1.12c 1.15cd 1.21cd 1.17c 1.15d 1.14cd 0.087 Liver 1.07 1.17 1.07 1.09 1.05 1.01 0.18 Bone 14.2 14.4 14.6 15.4 14.1 15.1 0.63 Micro elements, (mg/kg) Al Brain 43.1cd 52.0d 33.9c 50.4d 47.5d 48.2d 4.10 Heart 6.9cd 5.1def 6.1cdef 7.4c 7.0cd 4.5ef 0.79 Kidney 7.2cd 9.4c 7.1cd 7.5cd 5.4d 5.4d 1.03 Liver 15.4c 22.3cd 16.7c 20.9cd 25.3d 18.5cd 2.75 Cu Heart 8.7 12.7 9.8 14.4 10.3 7.4 2.07 Kidney 38.0 37.3 46.9 36.1 31.2 27.9 9.79 Liver 4,090 4,570 3,080 3,930 3,270 3,900 713 Fe Heart 152 143 154 146 162 134 10.9 Kidney 443 442 425 447 434 434 66.4 Liver 212cd 141d 208cd 162cd 262c 226c 27.8 Mn Heart 1.6cd 1.4d 1.80c 1.31d 1.57cd 1.50cd 0.13 Kidney 18.3cd 23.2cd 16.4c 23.2d 23.9cd 18.4cd 2.4 Liver 12.8 11.4 14.1 10.2 11.9 11.4 2.0 Se Kidney 1.1 1.2 1.3 1.1 1.1 1.2 0.07 Zn Heart 71.5 63.6 65.7 69.1 59.8 61.2 6.65 Kidney 83.9 110 120 110 85.1 95.2 15.0 Liver 48.5 48.4 44.8 43.5 36.8 46.3 6.91 aData represent least squares means; n = 5, 5, 7, 7, 5, and 6 for the control and treatments 1-5, respectively bDietary treatments were created by additions to a corn-SBM basal diet as follows: 1) (Control) 10% sand; 2) 9.3% sand + 0.7% AlCl3; 3) 7.5% sand, + 2.5% WTR; 4) 5% sand + 5%WTR; 5) 10% WTR; 6) 10% WTR + two times the added amount of mineral-vitamin premix + 1.29% dicalcium phosphate; Treatments 2 and 3 were formulated to contain 2,000 ppm Al, treatment 4 was formulated to contain 4,000 ppm Al, and treatments 5 and 6 were formulated to contain 8,000 ppm Al. cdefMeans within rows lacking a common superscript differ (P < 0.05).

PAGE 52

42 22.5-12.918.231.824.010.9-20.0-10.00.010.020.030.040.0123456TreatmentsApparent P absorption b b b b b a Figure 3-1 Effect of dietary Al source on apparent P absorption Dietary treatments were created by additions to a corn-SBM basal diet as follows: 1) (Control) 10% sand; 2) 9.3% sand + 0.7% AlCl3; 3) 7.5% sand, + 2.5% WTR; 4) 5% sand + 5%WTR; 5) 10% WTR; 6) 10% WTR + two times the added amount of mineral-vitamin premix + 1.29% dicalcium phosphate; Treatments 2 and 3 were formulated to contain 2,000 ppm Al, treatment 4 was formulated to contain 4,000 ppm Al, and treatments 5 and 6 were formulated to contain 8,000 ppm Al. The SE for treatments is 8.23. abMeans lacking a common superscript differ (P < 0.05).

PAGE 53

CHAPTER 4 EFFECT OF DIETARY ALUMINUM FROM WATER TREATMENT RESIDUALS ON BONE DENSITY AND BONE MINERAL CONTENT OF FEEDER LAMBS Introduction Animal waste, which is often applied to grazing land, contains P that can remain in the A horizon of the soil profile and may lead to water pollution (Haustein et al., 2000). Aluminum (Al), if applied to the land, is thought to complex with P and to reduce the soluble P concentrations in animal waste (OConnor et al., 2002). This type of reduction in soluble P levels of animal waste products could result in significant decreases in pollution by commercial livestock operations. Previous studies suggest that the application of Al containing water treatment residuals (WTR), a byproduct of water purification, increases the soils capacity to bind and retain P (Elliott et al., 2002). The chemical mixture of soil and WTR has been shown to increase the retention value of soil P by several fold (Novak and Watts, 2004) and could result in a possible solution to environmental concerns associated with animal waste disposal. A major concern is that WTR, containing high amounts of Al, may adversely affect P utilization and bone deposition in grazing livestock that inadvertently consume WTR. Highly available dietary Al may create unabsorbable P complexes in the intestinal tract and, Al toxicosis is often observed as a P deficiency (Valdivia, 1977). Diets fed to sheep containing 0.29% P and 2,000 ppm Al via AlCl3 resulted in reduced bone density (Rosa et al., 1982). Additionally, high amounts of bioavailable Al can also negatively 43

PAGE 54

44 impact the status of Fe, Zn, and Mg in sheep (Rosa et al., 1982). The bioavailability of Al varies in different WTR, but is generally much lower than Al compounds such as AlCl3 (OConnor et al., 2002). The purpose of this study was to determine the effect of dietary Al as WTR and AlCl3 on bone mineral content (BMC) and bone density in feeder lambs. Materials and Methods Animals, Diets, and Management Forty-two, (30 wether and 12 female) five to eight-mo-old lambs, (22 Suffolk and 14 Suffolk-crosses) were utilized in a 14 wk experiment at the University of Florida Sheep Nutritional Unit located in Gainesville, Florida. The experiment was conducted from June 6th until September 25, 2004. The lambs weighed between 22 to 39 kg at d zero Prior to the experiment, lambs received an 8-way Clostridial vaccination given as injections of 2 mL four wks apart (Ultra Choice 8; Pfizer Animal Health, Exton, PA) and were dewormed, with two 1 mL doses with Ivermectin, two wks apart (Ivomec; Merial Ltd., Iselin, NJ). To prevent coccidiosis, amprolium (Corid 9.6%; Merial, Duluth GA) was used as an oral drench with lambs receiving 1 mL daily in a five d sequence. Corn-SBM basal diets were formulated to meet NRC (1985) requirements for CP, TDN, minerals and vitamins for lambs of this weight and age (Table 4-1). Lambs were fed the basal diet at 1200 to 1300 glamb-1d-1 during a 3 wk adjustment period and 1300 to 1600 glamb-1d-1 throughout the experiment. Lambs were stratified by sex and randomly assigned to six dietary treatments: 1) control (10% sand), 2) (9.7% sand and 0.3% AlCl3), 3) (2.5% WTR and 7.5% sand), 4) (5% WTR and 5% sand), 5) (10% WTR and 0% sand), and 6) (10% WTR, 0% sand, plus double the quantities of the mineral-vitamin premix, and 1.29% dicalcium phosphate). The WTR was analyzed to contain 7.8% Al on a DM basis. Ten percent of each diet was either sand, WTR or the

PAGE 55

45 combination of the two. The diet concentration of Al were 910, 2000, 2000, 4000, 8000, and 8000 ppm respectively for the six diets. Lambs were housed (seven to each pen) covered, in earth-floored wooden pens (24 sq. m), which were bedded with pine wood chips. Animals were group fed in open troughs until d 84, when animals were placed in individual raised metabolic crates. All lambs had access to salt and ad libitum water. Dietary treatments were offered ad libitum and DM intake was monitored daily. Diets were not reformulated during the study. Sample Collection and Analyses Radiographic photometry was used to estimate bone mineral content (BMC) at 28 d, 69 d and 109 d. For each lamb, the left dorsal/palmer, third metacarpal region of the leg was radiographed with the use of a portable x-ray machine, (Easymatic Super 325; Universal X-Ray Products, Chicago, IL). The machine was set at 97 pkv, 30 ma, and 0.067 sec. One cm below the nutrient foramen of the third metacarpal, a cross section of the cannon bone was compared to the standard using the image analyzer and BMC was estimated by photodensitometry. A ten-step Al wedge, taped to the cassette parallel to the third metacarpal, was used as a standard in estimating BMC. Radiographs were taken with a 91.5 cm distance between the x-ray machine and the cassette (Meakim et al., 1981; Ott et al., 1987). The films were processed with an auto-radiograph processing machine, with Kodak products, and by Kodak development procedures (Eastman Kodak Co., Rochester, NY). Radiographs were evaluated with a photometer (Photvolt Corp., New York, NY); percentage light transmissions (%T) were used to determine solid matter The radiographs were zeroed using the thinnest Al step that could be distinguished as a

PAGE 56

46 differing shade from the next ascending step. The BMC was evaluated 2 cm descending from the nutrient foramen. Bone diameter and medullar width were taken to the nearest 0.2 mm using a plastic transparent ruler (Meakim et al., 1981). Determination of the %T reading was then analyzed graphically using a logarithmic calculation. The width of the bone segment, 2 cm below the nutrient foramen, was compared to the visible segments of the Al step wedge. On d 111, all animals were sacrificed at a USDA inspected facility and bone from left leg was removed for bone mineral and bone density analysis. To prepare bone for P, Ca, and Mg analyses, bone removed the left dorsal/palmer, third metacarpal region of the leg, was skinned, immediately wrapped in 0.9% saline-soaked cheese cloth and frozen at 0 C. After thawing, bone was cut into 2 cm sections, 2 cm below the nutrient foramen, and marrow was carefully removed. Samples were rinsed in deionized water and blotted dry. Specific gravity procedures (Kit ME-40290; Mettler Instruments Corp., Hightstown, NJ) were used to determine bone density (g/cm) as described by Williams et al. (1990, 1991c). Bone samples were then dried at 105 C for 16 h extracted with an ether soxhlet apparatus for 48 h, air dried for 10 h, and then oven dried at 105C again for 16 h. Dry samples were weighed, and ashed in a muffle furnace at 600C for 8 h. All P samples were analyzed with colorimetric procedures (Harris and Popat, 1954), while Ca and Mg were analyzed by flame atomic absorption spectrophotometry (Perkin-Elmer Model 5000, Perkin-Elmer Corp., Norwalk, CT).

PAGE 57

47 Statistical Analysis Bone density and BMC data were subjected to the GLM procedure of SAS (SAS for Windows v9; SAS Inst., Inc. Cary, NC) in a completely randomized arrangement. Significance was declared at P < 0.05, and tendencies were recognized at P < 0.15. Results Radiograph BMC At d 28, lambs receiving 2,000 ppm Al from WTR tended (P = 0.07) to have greater bone density than lambs receiving 2,000 ppm Al as AlCl3 (Table 4-2). Likewise, lambs receiving 8,000 ppm Al as WTR tended (P = 0.11) to have denser bone then those receiving 2,000 ppm Al via AlCl3. Overall, bone density was unaffected (P = 0.51) by treatment at d 69, as only lambs receiving 4,000 ppm Al from WTR had bones which tended to be more dense than the controls. Near the end of the study, (d 109) bone density was again unaffected by dietary Al content. Only bones from lambs receiving 8,000 ppm Al as WTR tended to be more dense (P = 0.15) than the controls, and no other difference or tendencies were observed. Bone Density via Specific Gravity Bone density as determined by specific gravity (Williams et al., 1990; 1991) was unaffected by treatment (P = 0.43). Bone density ranged from 1.88 to 1.94 g/cm3 and bones from lambs receiving 2,000 ppm Al as WTR tended to be more dense (P = 0.07) than bones from lambs receiving 4,000 ppm Al from WTR. Bone Mineral Analyses Bone mineral percentages (Table 4-3) of P and Ca were unaffected by treatment. Bone mineral percentages of Ca were also unaffected by treatment (P = 0.87). Bone Mg content differed (P < 0.05) in the lambs receiving 8,000 ppm from WTR plus double the

PAGE 58

48 minerals and vitamins, having lower bone deposits of Mg than those fed the control, 2,000 ppm Al via WTR, or 4,000 ppm Al via WTR (P < 0.05). Additionally, lambs receiving 4,000 ppm Al from WTR tended to have higher bone Mg content than lambs fed 8,000 ppm Al from WTR (P = 0.06). Discussion Radiographing techniques and specific gravity measurements revealed that dietary Al content had no effect on BMC or bone density. Bone density and P deposition is expected to decline when additional Al is ingested according to research conducted by Valdiva et al. (1977) and Rosa et al.(1982). Diets formulated with high Al content (up to 2,000 ppm) in previous work with mallard ducks and chicks (Capdevielle et al., 1998) and rats caused a decline (GmezAlonso et al., 1996; Zafar et al., 2004) in bone mineral declines with Al dietary additions. Yet, contradictory results have been described in much of the research conducted with ruminant species ( Valdivia et al., 1978; 1982; Rosa et al., 1982) Valdivia (1982) concluded that ruminants are less susceptible to toxic effects of Al than in other species. Normally, bone ash is 17.6 % P, and 37.7 % Ca (McDowell, 2003). Studies conducted by Validiva et al. (1982) showed bone mineral percentages of P that were slightly below average, 14 to 15%, as is seen in our study. However in the present study, differences were not observed among dietary treatments. Since differences were not observed, there was no notable effect of dietary Al intake on bone mineral deposition. The long bones of the appendages are often the last affected by P deficiencies (Williams et al., 1991a,b,c; McDowell, 2003). This could be a logical justification for the lack of treatment effect in the present study. A secondary explanation is that the dietary levels of P (0.25%), as seen in intake studies by Rosa et al. (1982), were high enough to compensate for the Al additions to the basal diet. Rosa et al. (1982) also

PAGE 59

49 reported that bone ash Ca levels were unaffected by dietary Al and ranged from 35 to 36 %, similar to results in our study which ranged from 33.1 to 39.9 % for bone ash Ca. Studies conducted with rodents (Cox and Dunn, 2001; Zafar et al., 2004) found that Ca deposits in the bone declined when dietary levels of Ca were deficient and Al was fed. In the present study, Ca levels were above the requirement and no declines in Ca bone deposits were observed. The absence of treatment effect is attributed to proper dietary Ca levels and Ca to P ratios. Summary and Conclusions A 111 d experiment was conducted to determine if the use of Al sources (AlCl3 vs. WTR) at various levels (910 to 8,000 ppm) affected BMC and bone density of growing sheep. Forty-two, 5 to 8-mo-old lambs, (12 ewe and 30 wethers) were utilized in a completely randomized experimental arrangement. Treatment, consisted of the following: 1) control (10% sand), 2) (9.7% sand and 0.3% AlCl3), 3) (2.5% WTR and 7.5% sand), 4) (5% WTR and 5% sand), 5) (10% WTR and 0% sand), 6) (10% WTR, 0% sand, double the quantities of the mineral-vitamin premix, and 1.29% dicalcium phosphate). Basal diets met all requirements and contained 0.25% P. The lambs weighed between 22 to 39 kg at d zero and between 45.9 to 53.3 kg on d 111. The WTR contained 7.8% Al on a DM basis. Ten percent of each diet was either sand, WTR, AlCl3 or the combination of two. The resulting concentrations of Al were 910, 2000, 4000, and 8000 ppm, respectively, for the six diets. On d 28, 69, and 109, radiographs were taken. Mean bone densities from radiographs were similar among treatments (P = 0.30). At experimental termination, d 111, animals were sacrificed. The third metacarpal was then used for specific gravity procedures, and no differences were observed among treatments (P > 0.40). Overall,

PAGE 60

50 results indicate that Al in various forms and levels fed to growing sheep provided adequate amounts of P (0.25%) and other required dietary nutrients had no effect on bone density over a period of 79 d or on specific gravity calculations of bone density over 111 d. Implications When sheep received adequate dietary concentrations of P (0.25%), Al from AlCl3 or WTR had no effect on bone density or composition. In relation to bone development, the Al-WTR that contains 7.8% Al, which was implemented, is safe for consumption by sheep up to 10% of their total diet.

PAGE 61

51 Table 4-1. Diet composition (as-fed) and analyses for average (n=18) concentrations for macroand micro-elements for treatments Treatmentsa Ingredient (%,as fed) 1 2 3 4 5 6 Ground Corn 41.1 41.1 41.1 41.1 41.1 39.9 Soybean hulls 12.5 12.5 12.5 12.5 12.5 12.5 Wet molasses, unfortified 10.0 10.0 10.0 10.0 10.0 10.0 Cottonseed hulls 8.0 8.0 8.0 8.0 8.0 8.0 Corn gluten meal, 60% CP 5.5 5.5 5.5 5.5 5.5 5.5 Alfalfa meal, 17% CP 5.0 5.0 5.0 5.0 5.0 5.0 Vegetable oil (soybean) 4.0 4.0 4.0 4.0 4.0 4.0 Sandb 10.0 9.3 7.5 5.0 Water treatment residualc 2.5 5.0 10.0 10.0 Aluminum chloride 0.7 Salt 1.0 1.0 1.0 1.0 1. 1.0 Urea 1.6 1.6 1.6 1.6 1.6 1.6 Ground limestone 0.7 0.7 0.7 0.7 0.7 0.7 Ammonium chloride 0.5 0.5 0.5 0.5 0.5 0.5 Flowers of sulfur 0.01 0.01 0.01 0.01 0.01 0.01 Mineral-Vitamin-premixd 0.01 0.01 0.01 0.01 0.01 0.02 Dicalcium phosphate 1.3 Analyses (ppme) Ca 7,170 7,120 7,220 7,440 7,300 10,000 Mg 2,780 2,730 2,700 2,880 2,870 3,020 Na 4,060 3,240 3,030 3,000 3,410 3,110 K 4,180 5,210 3,960 4,560 4,440 4,380 P 2,520 2,490 2,550 2,480 2,460 5,020 Al 910 2,320 2,270 3,9710 7,860 7,790 Co 7 5 6 5 5 8 Cu 31 33 33 32 34 42 Fe 66 65 67 66 66 70 Mn 11 13 13 13 14 19 Zn 74 70 71 70 67 79 aDietary treatments were created by additions to a corn-SBM basal diet as follows: 1) (Control) 10% sand; 2) 9.3% sand + 0.7% AlCl3; 3) 7.5% sand, + 2.5% WTR; 4) 5% sand + 5%WTR; 5) 10% WTR; 6) 10% WTR + two times the added amount of mineral-vitamin premix + 1.29% dicalcium phosphate; Treatments 2 and 3 were formulated to contain 2,000 ppm Al, treatment 4 was formulated to contain 4,000 ppm Al, and treatments 5 and 6 were formulated to contain 8,000 ppm Al. b Sand contained 0.2 % Fe, 0.01 % Al, 0.09% Ca, 0.03 % Mg, 0.1 % P, 0.005 % Mn, 0.004 % Cu, and 0.001 % Zn. c Water Treatment Residuals contained 0.30 % Fe, 7.8 % Al, 0.11 % Ca, 0.024 % Mg, 0.3 % P, 0.004 % Mn, 0.73 % S, 0.006 % Cu, and 0.002 % Zn. dMineral-Vitamin-premix contained 8.0% Mg (as oxide), 0.70% Fe (as sulfate), 2.40% S (as sulfate), 1.9 % Cu (as sulfate), 6.0 % Mn (as oxide), 0.47 % I (as iodate), 0.075 % Se (as sodium selenite), 4.5 % Zn (as oxide and sulfate), 133,363.4 IU/kg Vitamin A supplement, 412,272.7 IU/kg Vitamin D3 supplement, 259.1 IU/kg Vitamin E supplement, rice mill byproduct, and stabilized fat as a vitamin carrier. eDry matter basis.

PAGE 62

52 Table 4-2. Effect of dietary Al concentration and source on bone density of feeder lambs as determined by radiographya Treatmentb,c 1 2 3 4 5 6 SE mm, Day 28 5.71 4.78 6.15 5.48 6.07 4.90 0.55 69 5.76 6.14 6.60 6.91 6.49 6.22 0.44 109 4.96 5.03 5.01 5.48 6.44 6.08 0.67 aData represent least squares means, and pooled SE; across treatments; n = 7 per treatment bDietary treatments were created by additions to a corn-SBM basal diet as follows: 1) (Control) 10% sand; 2) 9.3% sand + 0.7% AlCl3; 3) 7.5% sand, + 2.5% WTR; 4) 5% sand + 5%WTR; 5) 10% WTR; 6) 10% WTR + two times the added amount of mineral-vitamin premix + 1.29% dicalcium phosphate; Treatments 2 and 3 were formulated to contain 2,000 ppm Al, treatment 4 was formulated to contain 4,000 ppm Al, and treatments 5 and 6 were formulated to contain 8,000 ppm Al. cMeans within rows did not differ (P < 0.05). Table 4-3. Effect of dietary Al on dry fat-free bone mineral concentrations of Ca, P and Mg for experimental dietsa Treatmentb 1 2 3 4 5 6 SE g/cm3c Bone Density 1.89 1.91 1.94 1.89 1.93 1.93 0.003 mg /cm3c P 74.5 76.1 75.2 81.1 81.1 78.3 5.9 Ca 209 209 197 211 211 191 1.3 Mg 4.13 3.72 4.08 4.24 3.78 3.89 0.07 %d Ash 69.9 68.8 69.1 68.9 68.9 68.9 1.56 P 14.2 14.4 14.6 15.4 14.1 15.1 0.63 Ca 39.9 39.4 38.1 39.5 37.0 37.7 18.3 Mg 0.79e 0.70ef 0.79e 0.79e 0.73ef 0.61f 0.04 aData represent least squares means and pooled SE; n = 7 per treatment bDietary treatments were created by additions to a corn-SBM basal diet as follows: 1) (Control) 10% sand; 2) 9.3% sand + 0.7% AlCl3; 3) 7.5% sand, + 2.5% WTR; 4) 5% sand + 5%WTR; 5) 10% WTR; 6) 10% WTR + two times the added amount of mineral-vitamin premix + 1.29% dicalcium phosphate; Treatments 2 and 3 were formulated to contain 2,000 ppm Al, treatment 4 was formulated to contain 4,000 ppm Al, and treatments 5 and 6 were formulated to contain 8,000 ppm Al. cCalculated using fresh weights dCalculated using Ash weights efMeans within rows lacking a common superscript differ (P < 0.05).

PAGE 63

CHAPTER 5 SUMMARY AND CONCLUSIONS In many developed nations, concerns about repeated application of manure to land `has led to strict laws and regulations because of increased P levels in nearby water bodies. Contamination with P can occur in sandy soils because of P leaching into the ground water, and in slit or clay soils because of runoff of soluble P or erosion of P from soil manure into local bodies of water. When P enters the water ways causes algae growth is stimulated. When the algae die oxygen content of the water is decreased and leads to aquatic plant and animal death. A chemical reaction between Al and P binds soluble P making it unavailable as a pollutant. During the water treatment process, Al is added to bind small particles, aiding in the sedimentation processes, and resulting in the formation of water treatment residuals (WTR). Water treatment residuals contain a nonavailable form of Al known to reduce P runoff and leaching. Aluminum, when consumed in a bioavailable form, decreases growth, intake, and body weight, and depresses bone deposition in several livestock species. A major concern is that the Al in WTR, thought to be non-available, may negatively impact an animal when ingested. At the University of Florida, data were gathered to determine 1) if WTR are harmful if ingested in amounts between 2,000 to 8,000 ppm Al and 2) determine the availability of Al in WTR when compared to a control and a diet containing a bioavailable form of Al (AlCl3). A 111-d study was conducted to determine if declines in intake, BW, ADG, bone mineral content (BMC), bone density, plasma P, tissue P, and 53

PAGE 64

54 apparent P absorption were produced by the dietary administration of WTR to growing lambs. Lambs were stratified by sex and randomly assigned to six dietary treatments; 1) control (10% sand), 2) (9.7% sand and 0.3% AlCl3), 3) (2.5% WTR and 7.5% sand), 4) (5% WTR and 5% sand), 5) (10% WTR and 0% sand), and 6) (10% WTR, 0% sand, plus double the quantities of the mineral-vitamin premix, and 1.29% dicalcium phosphate). The WTR was analyzed to contain 7.8% Al on a DM basis. Ten percent of each diet was either sand, WTR or the combination of the two. The diet concentrations of Al were 0, 2000, 4000, and 8000 ppm, respectively, for the six diets. Body weight, intake, and ADG data were compared among three inclusion levels of Al as WTR, and one level of Al from AlCl3. Plasma samples and lamb weights were collected every 14 d. Fecal collection (to determine apparent P absorption) occurred between d 91 and d 105, and individual feeding occurred between d 91 and d 111. Samples of blood, brain, liver, kidney, heart, and bone were collected upon experimental termination. Lamb ADG, BW, and intakes were unaffected by dietary levels of WTR when compared to the control. However, lambs fed 2,000 ppm Al from AlCl3 had reduced growth and lower ADG (P < 0.05) than other treatments. Plasma P concentrations were unaffected by treatments at wk 0 or wk 2. The control consistently had higher P concentrations than most other treatments, and the WTR treatments generally had higher P concentrations than lambs given AlCl3. Between wk 6 and wk 14, plasma P concentrations began to increase after a decline at wk 6. Currently, there is no evidence to explain this finding but this could be attributed to bone mineral resorptions could have caused plasma P levels to increase and then stabilize.

PAGE 65

55 Tissues were analyzed for concentrations of Ca, P, Mg, Cu, Fe, Mn, Se, and Zn. Phosphorus concentrations were unchanged across treatments for all tissues and bone, except kidney, where the control had a higher concentration of P than the lambs given 8,000 ppm Al form WTR (P < 0.05). Bone deposits of Mg were lowest for lambs fed 8,000 ppm Al from WTR with double the added mineral-vitamin premix, and 1.29% dicalcium phosphate. All other bone mineral concentrations were unaffected by dietary treatment. Iron concentrations were highest in the liver of lambs feed 8,000 ppm Al from WTR and lowest in the controls (P < 0.05). Aluminum varied in most tissues, but brain is the primary repository for Al and is the focus of much research. Concentrations of Al in the brain were highest for animals receiving 2,000 ppm Al from AlCl3 and lowest lambs given 2,000 ppm Al from WTR (P < 0.05). Concentration, of Al increased when Al from WTR was given in amounts above 2,000 ppm. The accumulation of Al in the brain has not been shown to be a threat to the animal and cannot be critiqued without further research. On d 91, lambs were placed in metabolism crates; feed and feces were collected for the determination of apparent P absorption. No differences in apparent P absorption were observed among WTR treatments, however the lambs administered 2,000 Al via AlCl3 had reduced apparent absorption of P. We conclude that Al in WTR does not interfere with the apparent absorption of dietary P, but AlCl3 causes apparent fecal P absorption to decline. There were no BMC and bone density differences in long bones collected from lambs in any treatment. This lack of differences among treatments perhaps may be

PAGE 66

56 attributed to the short duration of the study and because that bone mineral resorption had not yet occurred in the long bones. Based on the data collected during this trial and from previous studies, it can be determined that 2,000 ppm Al via AlCl3 results in poor animal performance and P tissue and plasma P declines. However, intakes, BW and ADG of lambs receiving WTR in amounts from 2,000 ppm to 8,000 ppm Al did not differ from the control. Thus, WTR does not appear to negatively affect performance of growing sheep. The apparent P absorption data strengthens the idea that Al in WTR is less available to the animal then the Al in AlCl3. Apparent P absorption was not altered in lambs fed WTR, but animals fed 2,000 ppm AlCl3 were negatively impacted. Additionally, plasma P and tissue mineral levels, with the exception of brain Al, were not altered with the administration of Al from WTR. Under these experimental conditions, dietary administration of Al from WTR did not cause physiological tissue damages. Overall, it has been demonstrated that Al from WTR does not negatively impact a growing lambs health or performance and could be administered at levels as high as 8,000 ppm Al without causing detrimental effects. Additional research in other ruminant species should be conducted before data can be proper applied to species other than the ovine.

PAGE 67

57 APPENDIX TABLE DATA Table A-1. Effect of dietary Al concentr ation and source on ADG of feeder lambsa Treatmentb 1 2 3 4 5 6 SE ADG, g Wk 2 125cd 9.3c 107cd 235cd 344d 134cd 109 4 116d 4.6c 227d 97.3cd 272d 116cd 68.7 6 274c 50.6df 213cd 250ce -6.4ef -32.4f 64.9 11 257de 287d 570c 593c 480cd 463ce 91.5 14 366 361 244 208 207 269 69.7 aData represent least squares means; n = 7 per treatment bDietary treatments were created by additions to a corn-SBM basal diet as follows: 1) (Control) 10% sand; 2) 9.3% sand + 0.7% AlCl3; 3) 7.5% sand, + 2.5% WTR; 4) 5% sand + 5%WTR; 5) 10% WTR; 6) 10% WTR + two times the added amount of mineral-vitamin premix + 1.29% dicalcium phosphate; Treatments 2 and 3 were formulated to contain 2,000 ppm Al, treatment 4 was formulated to c ontain 4,000 ppm Al, and treatments 5 and 6 were formulated to contain 8,000 ppm Al. cdefMeans within rows lacking a common superscript differ ( P < 0.05).

PAGE 68

LITERATURE CITED Ammerman, C.B., R. Valdivia, I.V. Rosa, P.R. Henry, J.P. Feaster, and W.G. Blue. 1984. Effect of sand or soil as a dietary component on phosphorus utilization by sheep. J. Anim. Sci. 58:1093-1099. Basta, N.T., R.J. Zupancic, and E.A. Dayton. 2000. Evaluating soil tests to predict bermuda grass growth in drinking water treatment residuals with phosphorus fertilizer. J. Environ. Qual. 29:2007-2012. Brady, N.C., and Weil, R.R. 2002. The Nature and Properties of Soils. Pearson Education, Inc. Capdevielle, M.C., L.E. Hart, J. Goof, and C.G. Scanes.1998. Aluminum and acid effects on calcium and phosphorus metabolism in young growing chickens (Gallus gallus domesticus) and mallard ducks (Anas platyrhynchos). Arch. Environ. Contam. Toxicol. 35:82-88. Chappard, D., P. Insalaco, and M. Audran. 2003. Aluminum osteodystrophy and celiac disease. Calcif. Tissue. Int. 10:223-26. Cox, K.A. and M.A. Dunn. 2001. Aluminum toxicity alters the regulation of calbindinD28k protein and mRNA expression in chick intestine. J. of Nutr. 131:2007-2013. Dayton, E.A., and N.T. Basta. 2001 Characterization of drinking water treatment residual for use as a soil constituent. Water Environ. Res. 73:52-57. Dayton, E.A., N.T. Basta, C.A. Jakober, and J.A. Hattey. 2003. Using treatment residuals to reduce phosphorus in agriculture runoff. Am. Water Works Assoc. J.95:151-159. Elliott, H.A., G.A. OConnor, P. Lu, and S. Brinton. 2002. Influence of water treatment residuals on P solubility and leaching. J. Environ. Qual. 31:1362-1369. Farm Press. 2004. Right phosphorus management can cut vegetable costs, runoff. PRIMEDIA Business Magazine & Media Inc. Tampa, FL Feb. 7. p4. 58

PAGE 69

59 Federal Register. 2004. Agency information collection activities; submission to OMB for review and approval; comment request; national pollutant discharge elimination system (NPDES) compliance assessment/certification information (renewal), EPA ICR Number 1427.07, OMB Control Number 2040-0110. May 24. Vol. 69, no. 100. Fethiere, R., R.D. Miles, and R.H. Harms. 1990. Influence of synthetic sodium aluminosilicate on laying hens fed different phosphorus levels. Poult. Sci. 69:2195-2208. Field, A.C. and D. Purves. 1964. The intake of soil by grazing sheep. Proc. Nutr. Soc. 23:24-25. GmezAlonso,C. P. Menndez-Rodrguez, M.J. Virgs-Soriano, J.L. Fernndez-Martn, M.T. Ferndez-Coto, and J.B. Cannata-Anda. 1996. Aluminum-induced osteogensis in osteopenic rats with normal renal functions. Calcif. Tissue Int. 64:534-541. Harris, W.D. and P. Popat. 1954. Determination of phosphorus content of lipids. Amer. Oil Chem. Soc. J 31:124-126. Haustein, G.K., T.C. Daniel, D.M. Miller, P.A. Moore, Jr., and R.W. McNew. 2000. Aluminum-containing residuals influence high-phosphorus soils and runoff water quality. J. Environ. Qual. 29:1954-1959. Healy W.B. 1967. Ingestion of soil by sheep. Proc. New Zealand Soc. Anim. Prod. 27:109-115. Healy W.B. 1968. Ingestion of soil by dairy cows. New Zealand J. Agr. Res. 11:487-490. Huff, W.E., P.A. Moore Jr., J.M. Balog, G.R. Bayyari, and N.C. Rath. 1996. Evaluation of toxicity of aluminum in younger broiler chickens. Poult. Sci. 75:1359-1365. Ippolito, J.A., K.A. Barbarick, D.M. Heil, J.P. Chandler and E.F. Redente. 2003. Phosphorus retention mechanism of water treatment residuals. J. Environ. Qual. 32:1857-1864. Kleinman, P.J.A., A.N. Sharpley, B.G. Moyer, and G.F. Elwinger. 2002. Effect of mineral and manure phosphorus sources on runoff phosphorus. J Environ. Qual. 31:2026-2033. Lanyon, L.S. 1994. Dairy manure and plant nutrient management issues affecting water quality and the dairy industry. J. Dairy Sci. 77:1999-2007. Lorentzen, A. 2004. Environmental group says Iowa not enforcing laws. The Associated Press State and Local Wire, May 20.

PAGE 70

60 Mann, R.A., and B.S. Roberts. 2000. Smiths and Roberts Business Law. 11th Ed. West Legal Studies in Business, Thomson Learning, Cincinnati, OH. pp. 997-1002. Martin, L.C., A. J. Clifford, and A.D. Tillman. 1969. Studies on sodium bentonite in ruminant diets containing urea. J. Anim. Sci. 29:777-778. McDowell, L.R. 2003. Minerals in Animal and Human Nutrition. 2nd Ed. Elsevier Sci., Amsterdam. McDowell, L.R. 1997. Minerals for Grazing Ruminants in Tropical Regions. 3rd Ed. Bull. animal science department University of Florida, Gainesville. Meakim, D.W., E.A. Ott, R.L. Asquith and J.P. Feaster. 1981. Estimation of mineral content of the equine third metacarpal by radiographic photometry. J. Anim. Sci. 53:1019-1026. Meyer, D. 2000. Dairying and the environment. J. Dairy Sci. 83:1419-1427. Miles, P.H., N.S. Wilkinson, and L.R. McDowell. 2001. Analysis of mineral for animal nutrition research 3rd ed. University of Florida, Gainesville, FL. Miller W.J. 1983. Phosphorus-ruminant-nutritional requirements, biochemistry and metabolism. National Feed Ingredient Association's Mineral Ingredient Handbook. NFIA, West Des Moines, Iowa. pp.1-14. NRC (National Research Council)S. 1985. Nutrient Requirements of Domestic Animals. Nutrient Requirements of Sheep, 5th Ed. Natl. Acad. Sci. Washington DC. Novak, J.M. and D.W. Watts. 2004. Increasing the phosphorus sorption capacity of southeastern coastal plain soils using water treatment residuals. Soil Sci. 169:206-214. National Research Council. 2000. Watershed Management for Potable Water Supply. Natl. Acad. Press. Washington, DC. OConnor, G.A., H.A. Elliott, and P. Lu. 2002. Characterizing water treatment residuals for P retention. Soil Crop Sci. Soc. Florida Proc. p67-73. Ott, E.A., L.A. Lawrence, and C. Ice. 1987. Use of the image analyzer for radiographic photometric estimation of bone mineral content. Proc. 10th Equine Nutr. Physiol. Sym. Colorado State University. June 11-13. Penn, C.J. and J.T. Sims. 2002. Phosphorus forms in biosolids amended soils, and losses in runoff; effects of water treatment processes. J. Environ. Qual. 31:1349-1361. Powers, W.J. 2003. Keeping science in environmental regulations: role of the animal scientist. J. Dairy Sci. 86:1045-1051.

PAGE 71

61 Rengel, Z. 2004. Aluminum cycling in the soil-plant-animal-human continuum. BioMetals 17:669-689. Rosa, V., P.R. Henry, and C.B. Ammerman. 1982. Interrelationship of dietary phosphorus, aluminum and iron on performance and tissue mineral composition in lambs. J. Anim. Sci. 55:1231-1240. Rotz, C.A., A.N Sharpley, L.D. Satter, W.J Gburek, and M.A. Sanderson. 2002. Production and feeding strategies for phosphorus management on dairy farms. J. Dairy Sci. 85:31423153. Smith, D.R., P.A. Moore Jr, C.V. Maxwell, B.E. Haggar, and T.C. Daniel. 2004. Reducing phosphorus runoff from swine manure with dietary phytase and aluminum chloride. J Environ. Qual. 33:1048-1054. Soon, Y.K. and T.E. Bates. 1982. Extractability and solubility of phosphate in soils amended with chemically treated sewage sludges. Soil Sci. 134:89-96. Tomas, F.M., and M. Somers. 1974. Phosphorus homeostasis in sheep. I. Effect of ligation of parotid salivary ducts. Aust. J. Agric. Res. 25:475-483. Underwood, E.J. and N.F. Suttle. 1999. The Mineral Nutrition of Livestock. 3rd Ed. Midlothian, Wallingford, UK. US Environmental Protection Agency (USEPA). 1997. Animal Waste Disposal Issues: Office of Inspections, Washington. DC. US Environmental Protection Agency (USEPA). 2003. Ecological soil screening level for an interim final report. OERR, Washington DC. Valdivia, R. 1977. Effect of dietary aluminum on phosphorus utilization by ruminants. Ph.D. Dissertation, University of Florida, Gainesville. Valdivia, R., C.B. Ammerman, P.R. Henry, J.P. Feaster, and C.J. Wilcox. 1982. Effect of dietary aluminum and phosphorus on performance, phosphorus utilization and tissue mineral composition in sheep. J. Anim. Sci. 55:402410. Valdivia, R., C.B. Ammerman, C.J. Wilcox, and P.R. Henry. 1978. Effects of dietary aluminum on animal performance and tissue mineral levels in growing steers. J. Anim. Sci.47:1351-1360. Water Resources. 2005. The official government website of Greensboro NC; water treatment process. Greensboro, NC, http://www.greensboro-nc.gov/water/ supply/treatment.htm. Accessed: May 17, 2005. Williams, S.N., L.A. Lawrence, L.R. McDowell, and N.S. Wilkinson. 1991a. Criteria of evaluate bone mineral in cattle: II. Noninvasive techniques. J. Anim. Sci. 69: 1243-1254.

PAGE 72

62 Williams, S.N., L.R. McDowell, A.C. Warnick, L.A. Lawrence, and N.S. Wilkinson. 1992. Influence of dietary phosphorus level on growth and reproduction of growing beef heifers. Int. J. Anim.. Sci. 7:137-142. Williams, S.N., L.R. McDowell, A.C. Warnick, L.A. Lawrence, and N.S. Wilkinson. 1990. Dietary phosphorus concentrations related to breaking load and chemical bone properties in heifers. J. Dairy Sci. 73:1100-1106. Williams, S.N., L.R. McDowell, A.C. Warnick, N.S. Wilkinson, and L.A. Lawrence. 1991b. Phosphorus concentrations in blood, milk, feces, bone and select fluids and tissues of growing heifers as affected by dietary phosphorus. Liv. Res. for Rural Dev. 3:67-79. Williams, S.N., L.R. McDowell, A.C. Warnick, N.S. Wilkinson, and L.A. Lawrence. 1991c. Criteria of evaluate bone mineral in cattle: I. Effect of dietary phosphorus on chemical, physical, and mechanical properties. J. Anim. Sci. 69:1232-1242. Whetter, P.A., and D.E. Ullrey. 1978. Improved fluorometric method for determination of selenium. J Assoc. Off. Anal. Chem. 4:927-930. Zafar, T.A., D. Teegarden, C. Ashendel, M.A. Dunn, and C.M. Weaver. 2004. Aluminum negatively impacts calcium utilization and bone calcium-deficient rats. Nutr. Res. 24:243-259.

PAGE 73

63 BIOGRAPHICAL SKETCH Rachel Van Alstyne was born in Roches ter, NY, on March 4, 1979, to Fred and Andrea Van Alstyne. She was raised in the city of Rochester until the birth of her brother, Timothy. At age seven, Rachel and her family moved to the suburbs of Rochester, to the town of Fairport, NY. From the age of 16, she maintained se veral jobs providing enough monetary solidity to attain the educa tion she desired after high sc hool graduation. Rachel has always had an incorrigible desire to be near animals. With the exception of her employment at Bruggers Bagels as a shift mana ger, in all of her jobs she was able to surround herself with animals and/or wildlife. Rachels employment pursuits led her from veterinary hospitals to the Seneca Park Zoo in Rochester, where she worked as an animal care attendant better known as a zoo keeper." Rachel found it to be difficult during her first year as an undergraduate, since she did not have the agricultural background many of her classmates did, but she persevered, and maintained a near perfect GPA, proving that she was cut out for this lifestyle. She desired enhancement in the field of animal care, and she began her pursuit to be a veterinarian. During her junior year, Rachel decided that ve terinary college was not on the path for which she was best suited. She graduated from Cornell University with her bachelor's degree in animal science in 2002. Upon graduati on, she was undecided as to which field she desired to pursue in gradua te school, and began work. In 2003 Rachel applied and was accepted to a master's program at the University of Florida with Dr. Lee

PAGE 74

64 McDowell. After diligent days and evenings working in both the lab and barn, through hurricanes, lost power, sick lambs, and inad equate staffing she was able to receive her degree in 2005. While at the Univ ersity of Florida she obtai ned a second master's degree, concurrently, in management, from the Wa rrington College of Business. Unlike many students, Rachel was unable to take even one semester to herself without enrollment in classes. Her second degree in management from the college was both simulating and cumbersome, but she was able to finish bot h degrees within a pe riod of two years.


Permanent Link: http://ufdc.ufl.edu/UFE0011627/00001

Material Information

Title: Effects of Dietary Aluminum from Water Treatment Residuals on Phosphorus Status and Bone Density in Growing Lambs
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: UFE0011627:00001

Permanent Link: http://ufdc.ufl.edu/UFE0011627/00001

Material Information

Title: Effects of Dietary Aluminum from Water Treatment Residuals on Phosphorus Status and Bone Density in Growing Lambs
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: UFE0011627:00001


This item has the following downloads:


Full Text












EFFECTS OF DIETARY ALUMINUM FROM WATER TREATMENT RESIDUALS
ON PHOSPHORUS STATUS AND BONE DENSITY IN GROWING LAMBS













By

RACHEL VAN ALSTYNE


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


2005

































Copyright 2005

by

Rachel Van Alstyne


































Dedicated to family and friends who stood by me through my educational journey.















ACKNOWLEDGMENTS

The author wishes to extend sincere gratitude to Dr. Lee McDowell, Dr. George

O'Connor, and Dr. Lokenga Badinga for their service on her graduate committee.

Thanks are extended to Dr. McDowell for his support, patience, and encouragement

throughout this endeavor. Thanks are extended to Dr. O'Connor for his professional

wisdom and support regarding soil science. Thanks are extended Dr. Badinga for his

valuable insight and suggestions.

The author would like to thank Dr. Paul Davis for his assistance throughout the trial

and writing processes. His hard work, loyalty, love, and support as a friend, partner, and

colleague have been paramount to the success of the author during her life while

attending the University of Florida, in the publication of this thesis, and in the life they

seek together in the future.

Much appreciation is extended to Nancy Wilkinson, Jan Kivipelto, and Dr. Maria

Silveira for aid in laboratory analyses and data interpretation. Their support, time, and

assistance have been priceless. Thanks are given to Dr. Lori Warren, Steve Vargas,

Jessica Scott, Carlos Alosilla, Kathy Arriola, Eric Fugisaki, Tom Crawford, Jose

Aparicio, and Luis Echevarria, for their hard work and support during the trial.

Additionally, the author would like to thank her best friend and roommate, Karen

Fratangelo, for her support and kind regards during stressful times. Karen and the author

have been friends since the 6th grade and have been able to stay close and lean on each

other. Karen aided in diapering sheep, ended an ear, and shared a kind heart during hard









times. Though it may seem odd, the author would like to give her kind regards not only to

the humans who aided in her success, but also to the lambs that will always have a soft

spot in her heart. Last but not least, the author would like to thank her parents, brother,

and canine companion Vixen for their love and support throughout her education and

throughout life. She would never have had the perseverance and confidence that she does

without their love, loyalty, support, encouragement and respect.
















TABLE OF CONTENTS

page

A C K N O W L E D G M E N T S ................................................................................................. iv

LIST OF TABLES .............................. ..... .... .. ................. ....... viii

ABSTRACT .............. .......................................... ix

CHAPTER

1 INTRODUCTION ............... ................. ........... ................. ... .... 1

2 REVIEW OF LITERATURE ......................................................... .............. 4

Historical Significance of Phosphorus.................... ...... .......................... 4
R e q u ire m e n ts ................................................................................................................5
Phosphorus D eficiencies.................................................. ... ....6
Phosphorus M etabolism and Transport ................................. ............. .................. 8
Aluminum and Phosphorus Interactions.................................. ....................... 10
Pollution and Phosphorus Application to Land................................................... 14
Regulations .................. ............... ...................... .............. ........... 16
W TR and Environm ental U ses ........................................................ ............. 19

3 EFFECT OF ALUMINUM-WATER TREATMENT RESIDUALS ON
PERFORMANCE AND MINERAL STATUS OF FEEDER LAMB S.....................23

Introdu action ...................................... ................................................. 2 3
M materials and M ethods ....................................................................... ..................24
Anim als, D iets, and M anagem ent ............................................ ............... 24
Sample Collection, Preparation, and Analyses........................................25
Statistical A analysis ........................................ ................... .. .. ... 26
R e su lts ...........................................................................................2 7
D isc u ssio n ............................................................................................................. 2 9
Sum m ary and C onclu sions .............................................................. .....................36
Im p location s ........................................................................... 3 7









4 EFFECT OF DIETARY ALUMINUM FROM WATER TREATMENT
RESIDUALS ON BONE DENSITY AND BONE MINERAL CONTENT OF
FEEDER LAM BS ................................... .. .. ........ .. ............43

Introdu action ...................................... ................................................. 4 3
M materials and M ethods ....................................................................... ..................44
Anim als, D iets, and M anagem ent ............................................ ............... 44
Statistical A analysis ........................ ............ ................ ....... 47
R e su lts ................................................................................................... ........ . 4 7
Radiograph BM C............................................................................... 47
Bone Density via Specific Gravity .......................................... ...............47
B one M ineral A nalyses ............................................... ............................ 47
D isc u ssio n ............................................................................................................. 4 8
Sum m ary and C onclu sions .............................................................. .....................49
Im p licatio n s ................................................................5 0

5 SUMMARY AND CONCLUSIONS.......................................................................53

APPEND IX : TABLE D A TA ............................................... ...... ......................... 57

L IT E R A T U R E C IT E D ............................................................................ ....................58

B IO G R A PH IC A L SK E TCH ..................................................................... ..................63















LIST OF TABLES


Table Page

3-1 Diet composition (as-fed) and analyses for average (n=18) concentrations for
macro- and micro-elements for treatments.................................... ............... 38

3-2 Effects of dietary Al concentration and source on BW of feeder lambs .................39

3-3 Effect of dietary Al concentration and source on feed intake of feeder lambs .......39

3-4 Effect of dietary Al concentration and source on plasma P of feeder lambs ..........40

3-5 Tissue mineral composition resulting from experimental diets ...........................41

4-1 Diet composition (as-fed) and analyses for average (n=18) concentrations for
macro- and micro-elements for treatments ......... ..................... .... ............51

4-2 Effect of dietary Al concentration and source on bone density of feeder lambs as
determ ined by radiography ............................................ ............................. 52

4-3 Effect of dietary Al on dry fat-free bone mineral concentrations of Ca, P and Mg
for experim mental diets ...................... ................ ............................52

A-1 Effect of dietary Al concentration and source on ADG of feeder lambs ................57















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

EFFECTS OF DIETARY ALUMINUM FROM WATER TREATMENT RESIDUALS
ON PHOSPHORUS STATUS AND BONE DENSITY IN GROWING LAMBS

By

Rachel Van Alstyne

August 2005

Chair: Lee Russell McDowell
Major Department: Animal Sciences

Experiments using growing feeder lambs were conducted to gather data on 1) the

safety of a Al-water treatment residual (WTR) ingested in amounts to provide between

2,000 and 8,000 ppm Al, and 2) the bioavailability of Al in WTR when compared to a

control (910 ppm Al from sand) and a diet containing a known bioavailable form of Al

from AlC13.

The study was conducted to examine changes in performance (ADG, BW, and feed

intake), tissue mineral concentrations, plasma P concentrations, bone mineral content

(BMC), bone density, and apparent P absorption. At experimental termination, samples

of brain, liver, kidney, heart, and bone were collected and analyzed for concentrations of

Ca, P, Mg, Cu, Fe, Mn, Se, and Zn. Thirty-two wether and ten female lambs were

assigned to six dietary treatments: 1) control (10% sand), 2) (9.7% sand and 0.3% AlC13),

3) (2.5% WTR and 7.5% sand), 4) (5% WTR and 5% sand), 5) (10% WTR and 0% sand),

and 6) (10% WTR, 0% sand, plus double the quantities of the mineral-vitamin premix,









and 1.29% dicalcium phosphate). Treatments 1-5 contained P at 0.25% and

concentrations of Al were 910, 2000, 4000, 8000 and 8000 ppm, respectively for the six

diets. Compared to the control, ADG, BW, and intakes were unaffected by dietary levels

of WTR (P > 0.05); however lambs fed 2,000 ppm Al from AlC13 had reduced body

weights and lower ADG (P < 0.05). The control, most often, had the highest plasma P

concentrations and the WTR treatments generally had higher P concentrations than lambs

given AlC13. During wk 6, plasma P concentrations declined for all animals but steadily

increased thereafter. Kidney P differed; control lambs had larger deposits of P than lambs

given 8,000 ppm Al from WTR (P < 0.05). Iron deposits were highest in livers from

lambs fed 8,000 ppm Al from WTR and lowest in the controls (P < 0.05). Brain Al was

highest for animals receiving 2,000 ppm Al from AlC13 and lowest for lambs given 2,000

ppm Al from WTR (P < 0.05). Brain Al concentrations increased when Al from WTR

was given in amounts above 2,000 ppm. Apparent P absorption did not differ among

WTR treatments and the control (range from 11 to 32 %), but lambs fed 2,000 Al via

A1C13 had a negative (-13%) apparent absorption of P. Values of BMC and bone density

did not vary with treatments; this is likely due to the short duration of the study. This

study found no evidence of health related defects because of the administration of the

WTR. The Al as AlC13 was more bioavailable with regard to plasma P levels and

performance, than Al via WTR; animals which were given the AlC13 were negatively

affected.














CHAPTER 1
INTRODUCTION

Manure transportation for refuse is costly. This results in the primary method for

animal waste disposal being application nearby land. Repeated long-term manure land

application leads to accumulation of phosphorus (P) (Novak and Watts, 2004). In the

United States, the livestock industry produces 500 million tons of manure each year

(Lorentzen, 2004) and many coastal soils have already become saturated with P (Novak

and Watts, 2004).

The majority of the P produced as animal waste is not adequately used for plant

uptake and much of the soil used by large industrial agriculture companies has reached its

maximum capacity for P adsorption (Novak and Watts, 2004). When manure is applied

to the land and the P remains stagnant on the upper crust of the soil bed it may be washed

away with heavy rains (Haustein et al., 2000; Federal Registar, 2004).

Phosphorus lost in leaching and runoff can lead to eutrophication, causing the

overgrowth of algae, and decreasing the survival of aquatic plants and animals (Novak

and Watts, 2004). These algae lead to a reduction in the oxygen levels within the water

and result in an overgrowth of anaerobic bacteria that generate toxins such as Pfissteria

which may result in death, rashes, respiratory illness, and memory loss in people and

animals (Haustein et al., 2000). The death and decomposition of aquatic plants can lead

to depressed aquatic oxygen levels, resulting in fish mortalities.

Aluminum (Al) applied to the land has been shown to reduce the soluble P

concentrations from animal waste (O'Connor et al., 2002). Aluminum salts have been









used to help minimize the amount of P released from animal feces. The chemical

reaction which occurs between the Al from salts and the P in the manure, result in a

decrease in P loss. This method has proved to be effective but it is costly (O'Connor et

al., 2002). The reduction of available P levels from animal waste products could result in

significant decreases in leaching and runoff, lessening contamination of water supplies.

Studies conducted at the University of Florida suggest that water treatment residuals

(WTR), especially those containing Al, increase the soils capacity to retain P. Water

treatment residuals bind P, lessening its availability and decreasing water pollution

caused by runoff (Elliott et al., 2002). Water treatment residuals are derived from the

water purification processes and can vary in their mineral content and P absorption

capacities, depending on the chemical used by the water treatment facility and the age or

dryness of the WTR. Thus, WTR can be high in Al, Fe or Ca oxides and upon drying or

aging become safe for land application (O'Connor et al., 2001; Dayton et al, 2003). The

WTR used throughout this experiment (including references, unless otherwise stated) are

high in Al-oxide from a source known for its high P sobbing capacity and will be

referenced as WTR. Water treatment residuals are the solid sediments that result after

raw water is coagulated, leaving behind amorphous Al oxides (Basta et al., 2000; Dayton

and Basta, 2001). These WTR contain amorphous solids that vary in size and shape.

Most WTR look like, and have the texture of a dark soil, but have little or no nutritive

value. However, WTR usually contain 4 to 8% nonavailable Al. In general, WTR are

discarded in landfills or in waterways, but both methods of disposal are costly and may

increase the price of drinking water (Novak et al., 2004).









Application of WTR to the land may result in one solution for animal waste

discard, while eliminating the burden and expense of WTR disposal. The chemical

mixture of soil and WTR has been proven to increase the retention value of soil P by

several fold (Novak et al., 2004).

One concern posed is that WTR contains high amounts of Al leading to

contamination and ecological risk for grazing animals, wildlife, surrounding floriculture

and water systems (USEPA, 2003). High levels of Al can adversely affect P utilization

and bone deposition. Aluminum toxicity is often observed as a P deficiency resulting in

bone density impairment (Valdivia, 1977). Toxicity is primarily linked to the degree of

Al bioavailability. In WTR, the bioavailability of Al varies, but is generally low

(O'Connor et al., 2002). During grazing, ruminants naturally consume up to 10% to 15%

of their total dry matter (DM) intake as soil (Field and Purves, 1964), and soil can contain

as much as 10% Al (Valdivia, 1977). Research with livestock at the University of

Florida demonstrated that increases in dietary Al decreased voluntary feed intake, and

feed efficiency, depressed P serum concentrations, and depressed growth and gains

(Valdivia, 1977; Rosa et al. 1982). In this research Al-WTR were directly fed to sheep to

simulate grazing-like conditions and soil consumption to emulate an ingestion of soil

material in amounts of 10% of their diet, for hypothetical assessment of health related

affects if inadvertent consumption of WTR was to occur. The following experiments

compared the bioavailability of Al from WTR to an available source of Al, in AlC13. The

main focus will be on the effects of these Al sources on P status in sheep.














CHAPTER 2
REVIEW OF LITERATURE

Historical Significance of Phosphorus

Phosphorus (P) and calium (Ca) are the two primary minerals that constitute bone

matter and are actively involved in bone development. Together, Ca and P are the most

abundant minerals in an animal's body (Miller, 1983). Eighty to 85% of an animal's P is

found in the bones and teeth. Combined, Ca and P make up 70% of the minerals found in

the body (McDowell, 2003).

The essentiality of P in bone development has been known since 1769, when bone

ash was analyzed, and P was found to be a primary component of bone material

(McDowell, 2003). Much of the early research on P was instituted in areas of South

Africa where deficiencies had become a growing concern. Low P diets had been linked

to lamsiekte and botulism in much of the grazing livestock throughout the continent of

Africa. Clinical signs which appeared included: bone chewing, depressed growth,

failures in reproduction, and reduced feed intake. Chewing the bones of dead carcasses is

the most significant indicator of a low P. The P deficiency forces the animal to find any

source of P, but the ingestion of the bones can also lead to consumption of Clostridium

botulinum and death (McDowell, 2003). In areas of Piaui, Brazil, 20,000 to 30,000 cattle

die yearly because of botulism. Today, low P diets and associated diseases are

problematic in tropical regions of the world. In Latin America, 73% of all forages

evaluated in a feed table publication were P deficient (McDowell, 1997).









Requirements

Phosphorus makes up 0.12% of the earths crust by volume but is not often found in

an uncomplexed form. Phosphorus is extremely reactive and is most commonly found as

phosphate in sedimentary rock deposits (McDowell, 2003). Requirements for P vary

depending on age, sex, activity, bioavailability of P, protein and energy in the feed, stress,

interactions between feed ingredients and nutrients, digestive anatomy and reproduction

status of the animal (Miller, 1983). Ruminant animals have a lower requirement for P

and Ca than carnivores and omnivores. For strict carnivores such as felines, the

requirement for P is 0. 60% and 0.80% for Ca, non-lactating adult humans require 0.70 to

1.25% P and 0.70 to 0.90% Ca while sheep require 0.16 to 0.38% of dietary dry matter

(DM) P and from 0.20 to 0.82% of DM Ca (NRC, 1985). The NRC (1985) estimated

endogenous loses of P, using a factorial method, to range from 20 mg/kg body weight at

maintenance to 30 mg/kg body weight in growing lambs. For proper absorption, P must

be present in a bioavailable form. Ruminants, unlike mongastrics, are able to use phytin

P from plants and it is considered to be available. Only about one third of P in most

plants is available to nonruminants (McDowell, 2003). Incomplete uptake can be linked

to an unavailable chemical form of the mineral in the plant, physical barriers in the plant

wall, or antagonist elements such as oxalic acid and phytic acid which can bind P, Ca, Fe,

Mn, and Zn (McDowell, 1997; 2003).

The amount of Ca and P found in feedstuffs varies among sources. The Ca: P ratio

in legumes is between 6:1 and 10:1 and is considered to be extremely low in P. Grasses,

if mature, are often low in both Ca and P. The values depend on soil conditions and plant

species. Alkaline soils are more abundant in trace minerals than acidic and sandy soils.

Tropical soils are usually older and acidic, marked by leaching and high environmental









temperature with compromised mineral contents, both in the soil and plants (McDowell,

1997). Seeds and seed by-products are rich in P, whereas animal by-products, tankage,

and milled flours are rich in both Ca and P (NRC, 1985). Animals on pasture will

develop P deficiencies before those fed high concentrate diets, as grains are high in P

(McDowell, 2003).

Many factors can influence absorption of minerals such as age, diet, parasites,

environmental stresses, disease, and toxic constituents. Growing animals naturally have

higher requirements for Ca and P because of bone development. A high protein and

energy diet increases the need for both Ca and P, but also increases the ability of the

animal to retain these minerals. During the rainy season in areas of South Africa and

South America, incidences of P deficiencies are common because more lush forages are

being consumed. The increased intake of energy and protein rich grasses increases

mineral requirements. Without supplementation, the forages are unable to provide many

of the needed minerals in sufficient amounts (McDowell, 1997). Infections and parasites

can affect the uptake of P and Ca. Nematodes have been proven to cause

demineralization of bone tissues in sheep (Underwood and Suttle, 1999). Stress and

activity of the animal will influence mineral needs, those under more stress or those with

higher physiological need, including growth, pregnancy, and lactation have the greatest

mineral requirements (McDowell, 2003).

Phosphorus Deficiencies

The most prevalent mineral element deficiency for grazing animals worldwide is

lack of P. The requirements for P and, frequently, other minerals are often not met by

grazing ruminants, and supplementation is often required. Additionally, certain elements

found in low pH tropical soils, such as Fe and Al, can hinder P absorption in the animal.









Deficiencies in P for grazing ruminants have been reported in 46 tropical countries in

Latin America, Southeast Asia, and Africa. The soil and forages in these livestock-

grazing areas of tropical countries are low in P (McDowell, 2003).

Phosphorus deficiency is most often seen in cattle and other grazing ruminants.

Young grasses may contain 0.3% P, but mature forages may contain 0.15% P or less

(McDowell, 1997; 2003). When dietary P becomes low, an early physiological response

is a decline in inorganic plasma P. Normal plasma P levels in ruminants are between 4.5

and 6 mg/100 ml. Levels below 4.5 mg/100 ml in ruminants are considered deficient. A

normal P level in ovine whole blood is between 35 to 45 mg and in plasma is between 4

to 9 mg per 100 ml, both of which will vary with age, and sex. Anorexic conditions are

first to occur with declines in P, decreasing feed efficiency and slowing energy

metabolism in ruminants, which ultimately results in a decline in growth (McDowell,

2003). Dry matter intake was reduced 40% in lambs receiving low-P diets and DM

digestibility was less than in animal offered the high-P diets (McDowell, 2003). A

significant decline in mineral P concentrations reduces the ability of animals to properly

digest fiber, protein and carry out normal metabolic functions (Miller, 1983).

Reproductive status may be compromised, primarily in females, which is often the most

economically damaging aspect of production. Animals deficient in P have been known to

go two to three years without calving (McDowell, 1997). Ruminants deficient in P are

listless, with swollen joints, abnormal stance, lameness, and have rough, dry hair coats.

Deficiency of P or Ca is similar to that of a deficiency in vitamin D, and lack of any of

these nutrients leads to rickets. Clinical signs can include weak bones, which may

become curved, enlarged hocks and joints, dragging of hind legs, beaded ribs and









deformed thorax (McDowell, 2003). Bone density decreases and the bone matrix

becomes soft and porous. With use of a noninvasive dual photon absorptiometry

technique, Williams et al. (1990) determined that dietary levels of 0.12 to 0.13% P lead to

bone demineralization in Angus heifers. Prior to bone disruptions, an animal suffers

stunted growth, depressed appetite, and weight loss. If the skeletal system is affected, the

bones (including: ribs, vertebrae, sternum, and spongy bone material) demineralizes

quickly. The last bones to be affected are the long bones and the smaller bones of

extremities. When P is deficient, even during normal activities, the bones can bend and

fracture, (McDowell, 2003).

Phosphorus Metabolism and Transport

Calcium and P regulation occurs as a result of the hormones, 1, 25 dihydroxy

cholecalciferol, parathyroid hormone and calcitonin. Regulation of normal Ca and P

levels depend on bodily excretion, bone deposition, resorption, and intestinal absorption

(Miller, 1983).

Phosphorus and proper availability of P depends on the Ca to P ratio, of which

should be between 1:1 or 2:1 for most monogastic species. However, for ruminants,

ratios below 1:1 and over 7:1 will negatively effect growth and feed intake. If Ca and P

needs are not met, tetany will occur as the animal withdraws Ca and P from bone in order

to maintain normal blood concentrations. The status of vitamin D is important to obtain a

desirable Ca:P ratio (McDowell, 1997; 2003). Over time, if Ca and P concentrations are

low, bone becomes soft and bone density is impaired. Most of the Ca and P in bone is in

the form of calcium phosphate and hydroxyapatite. The exact make up of bone material

varies with age, sex, physical activities, and reproductive status, but consistency is seen

within species and their stages of life (McDowell, 2003).









Absorption of P in ruminants occurs throughout the intestinal tract, including the

rumen, but is optimized in the small intestine. Its uptake occurs through active and

passive diffusion and is dependent on the solubility of the membranes that it comes in

contact with. Absorption is favored when the mineral is held in solution. Factors which

effect uptake of P include: digestive system pH, age, parasites, and other mineral intakes,

particularly Ca and Al (McDowell, 1997; 2003). Large amounts of bioavailable Al form

insoluble phosphates which bind P, making it unavailable to the animal (McDowell,

2003).

Bone undergoes turnover daily and in turn affects the P plasma levels of the animal.

Osteoblasts cause new bone formation, while osteoclasts (large multinucleated cells)

reabsorb the bone tissue. Most of the nonskeletal portion of P is found in the red blood

cells, muscle tissue and nervous system. Much of this P is used to regulate oxygen and

hemoglobin in the blood. Status of P in the body can be estimated by plasma or fecal

excretion. Feces is the primary pathway for P excretion in ruminants and non-

carnivorous animals. Carnivores excrete more P in the urine over that in the feces. In

diets low in P, the body naturally conserves P, particularly in herbivores and little to no P

is excreted in the urine (McDowell, 2003).

Phosphorus is used in almost every metabolic system, including those of ruminal

microorganisms, digestion, appetite simulation, feed conversion, fatty acid transport,

metabolism of nucleoproteins, maintenance of active cells, enzymes, hormones, and for

milk, egg, and muscle synthesis (Miller, 1983; McDowell, 1997; 2003).

Evaluation of P status can be determined by the concentration in bone, since the

majority of the mineral is in bone. Heifers fed low (0.12%) P diets at had a much lower









cortical bone index, medial lateral wall thickness, breaking load, and total ash than those

receiving a 0.20% P diet (Williams et al., 1991b). Bone density of the ribs and vertebrae

was also affected by P status (Williams et al., 1990). Blood, bone, feces, rumen fluid and

saliva can all be used with various degrees of success to indicate P status of a ruminant

and reflected dietary P levels (Williams et al., 1991a,c).

Aluminum and Phosphorus Interactions

Aluminum is the third most abundant element in the earth's crust, following silicon

and oxygen, and is the most common metal found in the earth's crust (O'Connor et al.,

2002). Aluminum is highly reactive and does not normally appear in its elemental form;

instead, Al binds to other elements or compounds (McDowell, 2003). Soil Al

concentrations can range from 1 to 30% but are typically in the 0.5 to 10% range by

weight (O'Connor et al., 2002). It is not uncommon to find high amounts of Al

complexes in tropical sandy soils, binding soil P and making it unavailable for plant

uptake (McDowell, 2003).

Aluminum chloride (AlC13) was added to fields of manure covered soil and reduced

P runoff by 53% (Smith et al., 2004). Data such as this prove that Al can bind P and

increase a soils P sorption capability.

In almost all cases, Al is considered to be a toxic mineral, and is not considered to

be a required element, except possibly in female rodents (McDowell, 2003). Rosa et al.

(1982) reported that increases in dietary P in sheep increased feed intake while it was

decreased by increases in Al and Fe. Increased dietary levels of Fe and Al in sheep diets

resulted in weight losses; ADG was decreased from 156 to 97g/d in high Fe diets and

from 159 to 95g/d in high Al diets. When additional P was added to diets containing high

Fe or Al, ADG losses were minimized. The rationale for this response was that the diet









being fed was borderline to deficient in P either at 0.17% to 0.23 % (NRC, 1985).

Additionally, plasma P levels increased with Fe and decreased with Al diets (Rosa et al.,

1982). Aluminum is not added to animal diets and, in most cases, is found in feeds only

because of contamination; whereas P is often added to animal feeds and mineral

mixtures.

When Al is absorbed via lungs, skin and intestines, only small amounts are actually

retained and can be reduced further with fluorine (F) consumption. Most Al is excreted

in the feces and urine (McDowell, 2003). In a study at the University of Florida, lambs

were given 2,000 ppm of an available source of Al (AlC13) and Al tissues levels were

only mildly elevated (Valdivia et al., 1982). In a similar study, calves were given 1,200

ppm of AlC13, and performance was not influenced and changes in tissue constituents

were only mildly elevated (Valdivia et al., 1978). The kidneys, liver, skeleton and brain

are often the tissues affected by Al toxicities (McDowell, 2003).

High dietary available Al can result in unabsorbable complexes with P in the

intestinal tract. The first effect from a low dietary P level is a decline in plasma P

(Williams et al., 1991a,c) further characteristics, including bone demineralization, then

follow (McDowell, 2003). When dietary Al exceeds the maximum tolerable level

suggested by the NRC (1985) of 1,000 ppm, animals develop characteristics of Al

toxicosis. Phosphorus is the mineral primarily affected when toxic levels of Al are

administered. An insoluble complex of Al and P is formed in the digestive system of the

animal, binding P and making it unavailable, as seen in sheep fed high levels of Al, and

signs ofP deficiency resulted (Valdivia, 1977). Bone ash and bone Mg level were

reduced when 1,450 ppm Al (chloride form) was given to wether lambs (Rosa et al.,









1982). Plasma P levels in sheep given 0.15% P with no added Al were 6.9 mg/100 ml

compared to 3.6mg/100ml for those receiving 2,000 ppm Al. Lambs fed 2,000 ppm Al

also had lower gains and feed intakes. All animals apparent P absorption was negatively

impacted except those fed high P with low Al concentrations. Correspondingly, plasma

Ca levels were reduced 0.24 mg/100 ml when 2,000 ppm Al was added. Non-ruminant

species are less tolerant to Al toxicity than ruminants. If the same studies were conducted

on monogastric animals, toxicosis would develop using 2,000 ppm Al and would

ultimately lead to death. The rational is that within the rumen Al complexes with organic

anions, not affecting P radicals in the same manner as a monogastric animal (Valdivia et

al., 1982).

High Al concentrations have also been linked to the possible onset of Alzheimer's

disease. No factual evidence has been documented to prove if an Al concentration in the

brain actually does affect the disease's occurrence. Through the influence of the disease

in the medical field is elastic and is currently being methodically investigated

(McDowell, 2003).

Toxicity is primarily linked to a high Al bioavailability (McDowell, 1997; 2003;

O'Connor, 2002). When Al toxicity is observed in the ruminant, bone density is often

impaired (Valdivia, 1977). Abnormally high amounts ofbioavailable Al can also impact

the status of Fe, Zn, and Mg in sheep. Dietary amounts of AlC13 at 1,000 ppm decreased

bone and kidney concentrations of Mg, additional antagonistic affects developed for P

and Ca as well. (Rosa et al., 1982). Bone ash was reported by Valdivia et al. (1978) to

contain lower amounts of Mg for animals fed diets containing AlC13.









The binding of P to WTR brings about concerns that plants will be limited in

required minerals such as P, Ca and Mg because they will become unavailable. Crop

yields could be negatively impacted if too much P is bound to Al or if increases in heavy

metal contents are realized within the soil (Novak and Watts, 2004). If P becomes

unavailable for plant uptake, deficiencies in both plants and animals could occur. Rosa et

al. (1982) concluded that excessive bioavailable dietary Al increases P requirements.

This may be particularly true when animals are grazing on acidic tropical pastures. In

acid soils, Al and Fe become more available and both complex with P and render it

unavailable to plants.

Acid soils with a pH of 5 or less usually contain higher amounts of available Al and

Fe (USEPA, 2003). Water treatment residuals have a pH above 5, which varies

somewhat from slightly acidic to moderately basic, and alkaline sources could act as

buffers to the soil. Past research has concluded that an elevated soil pH can be

maintained with long term use of some WTR, that have a low Al solubility. It is

unknown, but has been suggested, that WTR could have an opposite effect on the living

system and could lower the pH in the digestive systems of animals which consume it

directly (O'Connor et al., 2002). It is well known that, in general, soils with an alkaline

pH have higher mineral concentrations, than acidic soils. However, Fe, Co, Cu, Mn, and

Zn are much more available in acidic compared to alkaline soils (McDowell, 1997).

Many tropical soils are acidic (< 5.0 pH) with low P concentrations in forages. Acidic

conditions often result in high concentrations of Al and Fe which bind other minerals

(McDowell, 2003). In a study used to determine the affects of sand and soil ingestion in









sheep, tropical soils from Costa Rica with a pH of 5.2, were shown to negatively affect

the animals more than soils with higher pH's (Ammerman et al., 1984).

Pollution and Phosphorus Application to Land

Both the absolute number and percentages of the U.S. population employed strictly

in farming has fallen dramatically over time. The pressure to produce enough food, with

a smaller number of farmers, has had a worldwide impact on agricultural practices,

including the expansion of agricultural into marginal lands and the over use of land in

general. The agricultural industry needs to remain steadfast in providing adequate food

supplies, but we must not compromise environmental, socio-economic, human, and

wildlife health issues. In our effort to increase food production, pollution of our water

systems has become an issue of pressing attention. In many farming practices, manure

application to the land has become environmentally problematic. The majority of P

applied to the land as manure often is converted into an insoluble form in the surface

horizon of the soil. The accumulated P is subject to erosion or runoff following heavy

rains and transported to surface water. Thus, regulations on manure application rates

have developed to avoid P pollution of surface waters. (Dayton and Basta, 2001;

O'Connor et al., 2002; Dayton et al., 2003).

Animal producers oppose new stricter regulations placed on manure use because

the cost of compliance can be high. In Okeechobee County, Florida, the state has

mandated environmental improvements for certain farms. The state shared 75% of the

cost to update dairy facilities utilizing 456 employees and 50 million dollars (Lanyon,

1994). There are management methods that can be applied to decrease P runoff, but

many are expensive when applied to large farming operations. Using an intensive

management system on an average size farm of 100 head that was feeding a high quality









pasture, reduced concentrate feeding by 16%, and resulted in a 5% lower milk yield Yet,

this system also reduced the manure application to the land, lowered feed costs, and

reduced manure handling procedures so that the farm was able to increase overall annual

profitability by $10,000, which is equivalent to $93/cow (Rotz et al., 2002). The same

method was utilized by large farms, with 800 head, and profits were increased by

$23/cow, but only with an increase in milk production (Rotz et al., 2002).

It is a necessity to protect the land from erosion and the water from P pollution

caused by manure land application practices, but it is a struggle between the better of two

interests. A study in Pennsylvania researched several species of food animals to try to

determine differences among species and P production in manure. Three soils that

contained manure from ruminants, swine, and poultry were evaluated. Differences in P

concentrations among species could not attributed to any pertinent factor and could be

assumed to be a result of initial P variations, differences in the P distribution of the soils,

or the mixing of the soils and manures. Mixing of all manure types decreased P runoff

and was deemed useful in reducing P losses during heavy rains. Mixing the soil and

manure promotes sorption of P materials and dilutes the P in the soil surface (Kleinman

et al., 2002). Ideally, soil mixing could occur on farms, but labor and machinery costs

make the process unrealistic for large scale operations. The current strategies used to

reduce P runoff and leaching are soil tillage, crop residue management, cover crops,

buffer strips, contour tillage, runoff water impoundment and terracing. These techniques

have not be proven to achieve enough success to be used solely, or cooperatively to

reduce the current environmental problem (Dayton et al., 2003).









Regulations

Scrutiny from the general public and governmental agencies has developed with the

increasing pollutants detected in water bodies throughout the United States. In 1995, a

manure spill of 144 million liters, twice the size of the Exxon Valdez oil spill, occurred in

North Carolina (USEPA, 1997). Farms in the United States are being forced to adhere to

strict laws designed to protect the general public involving issues of odor control, water

and food safety (Powers, 2003; Federal Registar, 2004). In 1969, Congress passed the

National Environmental Policy Act (NEPA). The NEPA has two major divisions; the

Council of Environmental Quality (CEQ) and the Environmental Impact Agency (EIA).

The CEQ consists of a board of three members who advise the president on

environmental issues. The EIA oversees legislation proposed for federal action on

environmental issues (Mann and Roberts, 2000). Environmental law is governed by

statutory laws and is regulated by federal, state and local administrative agencies. The

Environmental Protection Agency (EPA) is the federal agency that oversees such issues,

(Mann and Roberts, 2000), having jurisdiction with 10 regional offices nation wide

(Meyer, 2000).

According to environmental research, the sheer amount of waste generated by large

animal facilities poses risk to ground and surface water (Lorentzen, 2004). According to

the EPA, farming creates 455 million metric tons of manure each year (Lorentzen, 2004).

In 1972, Congress amended the Federal Water Pollution Control Act (FWPCA) of 1948

with the Clean Water Act (CWA) of 1972 (Powers, 2003; Lorentzen, 2004). Again in

1977, 1981, 1987, and 2002, the CWA was amended to ensure clean water for the

following: recreational use, protection of the wildlife, and to eliminate pollutants into the

ground and drinking water. Concerns that embody the agricultural industry involve









leaching and runoff of nitrogen, solids, and P into the ground water, water ways and

water beds (Lorentzen, 2004).

Violators of the CWA are subject to both civil and criminal charges. Criminal

charges only apply if the violation was intentional. If charged criminally, the fines can

range from $2,500 to 1,000,000 dollars and from one to 15 years in prison. Civil charges

pertain to all other violations. Ignorance does not preclude one from dismissal of civil or

criminal charges. Civil fines can reach a limit of $10,000 a day and an overall maximum

of $25,000 per violation (Miller, 2004).

Watersheds do not always use filtration techniques when purifying natural water

sources (Rotz et al., 2002). A prime example is the New York State watershed located in

the Catskill Mountains. This particular region of the state is primarily covered with

forests and dairy farms, and supplies 4.5 billion liters of water to people in New York

City each day (NRC, 2000). The New York watershed which provides 90% of the

drinking water to the city, is purified only chemically, and serious harm could result if

manure solids were to contaminate the water systems (Rotz et al., 2002).

As defined by the CWA, there are two sources of pollution, point and non-point

sources. Point source means there is one defined place or confined area in which the

pollution has been released. Point source regulations mandate effluent limitations, based

on technological advancements, on the amount of pollution which can be discharged

from one source into a body of water. Concentrated animal feeding operations (CAFO'

s) are often considered point source pollution candidates (Meyer, 2000), and are defined

as operating with 700 cattle or a total of 1,000 animals (Lanyon, 1994). Non-point source

pollution occurs when the source of pollution can not be traced to a single area. Non-









point source is more often the cause of agricultural pollution; in regards to land use, run

off, and leaching (Mann and Roberts, 2000). Non-point pollution may not even be

observed in the watersheds that is directly affected, but may be carried for many meters

down stream and damage areas with no direct contact with the original pollutant (Lanyon,

1994). Classically, farms have been identified as non-point sources of pollution. It has

been predicted that within the near future, with increasing regulations and, because of

public agendas and concerns, smaller farms will too, be included in point source pollution

policies (Lanyon, 1994). For any type of discharge into open water ways, permits by the

National Pollutant Discharge Elimination System (NPDES) are required each time and

stricter rules are in the near future (Meyer, 2000).

Machine and equipment regulations governing businesses involving environmental

law are determined by the notion of best available control technology (BACT). This

requires that procedures and machines in use need to meet EPA standards for pollution-

control. New businesses need to follow standards more strictly than businesses already in

existence. As technology advances, new techniques develop that make it possible to

reduce pollution. New companies are legally bound to effectively alleviating pollution

with the use of advanced technologies. Timetables for existing companies have been

applied, meaning that the replacement of old equipment is to be implemented within a

reasonable time period. The replacement equipment protocol for existing companies

should then be based upon the best practical control technology (BPCT) law by replacing,

rather than repairing, equipment, to meet the most current EPA standards (Miller, 2004).

Many of the new regulations imposed on businesses regarding environmental

safety are locally mandated by state and county polices. In Maryland, Virginia, and









Delaware, stricter polices are being implemented in regards to P application to the land.

In Maryland, all P application must abide by the Water Quality Act of 1998, which

dictate soil testing to determine if the soil is saturated with P, and if manure application

can be permitted. In Virginia, food animal practices are closely scrutinized, particularly

the poultry industry; in Delaware manure can usually be treated once every three years to

comply with soil P limitations (Penn and Sims, 2002).

Water Quality Control Boards are now being mandated to more strictly adhere to

the monitoring of N and P levels in the soils. In California, there is an overabundance in

the pollutant count in several bodies of water of both P and N. Leaching and run off from

manure enriched fertilizers is thought to be the primary cause. The reality is that this

type of fertilization is a matter of convenience, availability, and cost profitability rather

than providing the optimal nutrients for the flora or concern for the ecosystem (Farm

Press, 2004).

WTR and Environmental Uses

Currently, there is no solution for the distribution of the large quantity of manure

produced in the livestock industry. The major issue at hand is the confinement of large

operations to small areas of land. Conflicts arrive in application and concentrations of

allowable feces. Both N and P are constituents of animal waste products, and are harmful

pollutants, yet federal, state, and county standards differ in the applicable uses and

concentrations of manure for land, resulting in confusion as to how manure should be

properly applied (Lanyon, 1994).

Water treatment residuals (WTR) are by-products from water purification

procedures. They are rich in metals like Al and Fe, though the exact composition can

vary. The elemental levels of Al, Fe, and Ca vary when comparing WTR depending on









the chemical used during the water treatment process and the age (or dryness) of the

WTR. In turn, these differences will reflect different abilities to adsorb P (Dayton et al.,

2003; Ippolito et al., 2003). During the water treatment process, a chemical, called a

coagulate, is added to the water and later forms WTR. This addition of chemicals to

water will cause a reaction and form a flocculent precipitate, which coats small particles,

such as clays making them more likely to be removed by sedimentation or filtration.

Aluminum sulfate (iron sulfate, or calcium sulfate) coagulates may be added to raw

water, (the WTR of interest for all further discussion is Al based). The water is then

circulated with vigor to uniformly disperse the Al product. Aluminum reacts readily with

alkaline products within the water and produces an Al hydroxide solid, which has

entrapped impurities. The sedimentation process allows the solids to settle-out. These

solid by-products are Al oxides bound to clay size particles and are known as WTR. The

processes of coagulation and sedimentation usually precedes filtration in a water

treatment plant, and serves to reduce turbidity and increase the efficiency of bacterial

removal by filtration (Dayton and Basta, 2001; Brady and Weil, 2002; Ippolito et al.,

2003; Water Resources, 2005). The physical characteristics of these WTR are similar to

top soils (Haustein et al., 2000).

The use of WTR and metal-binding by-products could be one solution to the

accumulation of soluble P in the top layer of soil, which leads to nonpoint pollution

during heavy rains (Penn and Sims, 2002). In particular, Al containing WTR would

benefit sandy soils low in organic material. Sandy soils tend to provide little P retention

capabilities and runoff is likely (Penn and Sims, 2002). Soils that are saturated with P

may also benefit from WTR application. It has been shown that P saturated soils are









unable to hold added P and thus will result in P ground water complications (Penn and

Sims, 2002). Added Al in the form of WTR may help depress P runoff by increasing soil

P retention capabilities (O'Connor et al., 2002; Penn and Sims, 2002). Publications in

2002 indicated a reduction in P leaching with the addition of Fe and Al from biosolids,

claiming that metal oxides formed lead to increased P retention (Soon and Bates, 2002).

Research at the University of Florida concluded that increases in dietary Al levels

reduced feed intake, gains and P plasma concentrations in sheep. The Al given to these

animals was in the form of AlC13. The impact of additional Al was not positive for

animal gains as ADG was 105 and 148 g/d for those consuming a high Al or a low Al

diet, respectively. When additional dietary P was given, the ADG increased, but it was

not as high as for animals not consuming any Al (Rosa et al., 1982). These results

demonstrate the capabilities Al had to lower P status in the animal, but it is unknown

what will occur if a less bioavailable form of Al is fed. Other mineral plasma

concentrations were also impaired with increased dietary Al. Magnesium content was

depressed in the kidneys, and bone of those animals receiving the high dietary Al (Rosa

et al., 1982). Similar results using Mg have also been documented at Rutgers University

in avian species. Young chicks and mallard ducks when fed high Al diets, as AlC13, had

a high incidence of P binding, lowered P serum levels, depressed growth, lowered tibia

weights and lower bone mineralization (Capdevielle et al., 1997).

Few studies have been conducted to determine the results of P accumulation,

ground water pollution, and the quantity of Al which is capable of binding P in WTR

when consumed by ruminants. The majority of studies in regards to WTR and Al content

have been focused on the ecological risks associated with plants in acidic soils






22


(O'Connor et al., 2002). Studies involving soil P binding mechanisms have also proven

to be helpful. Phosphorus absorption capacity was increased by 20 times with the use of

WTR when compared to high Al clay (Haustein et al., 2000).

Applications involving pollution control with the use of WTR fed to sheep will be

implemented here to compare the bioavailability of Al from WTR to an available source

of Al (AlC13) and evaluate how Al affects the performance of growing sheep.














CHAPTER 3
EFFECT OF ALUMINUM-WATER TREATMENT RESIDUALS ON
PERFORMANCE AND MINERAL STATUS OF FEEDER LAMBS

Introduction

Ingestion of highly available dietary Al (e.g. AlC13) by livestock may result in P

deficiency. Aluminum toxicity is often observed as a P deficiency (Valdivia, 1977).

Additionally, high amounts of bioavailable Al can also impact the status of Fe, Zn, and

Mg in sheep (Rosa et al., 1982).

Under grazing conditions, ruminants typically consume 10% to 15% of their DM

intake as soil (Field and Purves 1964; Healy, 1967; 1968). In sheep dietary Al

suppressed voluntary feed intake, feed efficiency, plasma P, growth, and gains (Rosa et

al., 1982). When additional P was ingested, these negative effects were less severe but

were still evident.

Water treatment residuals (WTR) are the byproducts from a water purification

procedure, and can contain high amounts of Al, Fe or Ca; here they contain high amounts

of Al and has a high P sorption capacity. The bioavailability of Al in WTR varies, but is

generally low and thought to be harmless (O'Connor et al., 2002). Since Al is highly

reactive and has been shown to chemically bind P, the administration of WTR on manure

containing soils could be a solution for P pollution of water systems by increasing soil P

retention capabilities (Penn and Sims, 2002). Concerns occur because of possible

ingestion of the WTR by grazing animals and the reaction of Al and P in a low pH









system. No previous research has been conducted to determine the potential toxicity of

WTR when directly consumed by grazing ruminants.

The purpose of this study was to determine if feeding growing lambs a bioavailable

source of Al (AlC13) versus a less available source of Al from a WTR would affect

growth, feed intake, plasma P levels, tissue concentrations, and apparent P absorption.

Materials and Methods

Animals, Diets, and Management

Forty-two, wether (30) and female (12), five to eight-mo-old lambs, (22 Suffolk

and 14 Suffolk-crosses) were utilized in a 111-d experiment at the University of Florida

Sheep Nutritional Unit located in Gainesville, Florida. The experiment was conducted

from June 6th until September 25, 2004. The lambs weighed between 22 to 39 kg at d

zero. Lambs were shorn on d 42 in an attempt to combat heat stress and to increase

optimum feed intake. Prior to the experiment, lambs were vaccinated with an 8-way

Clostridial given as an injection of 2-mL, four wks apart (Ultra Choice 8; Pfizer Animal

Health, Exton, PA) and were dewormed, with two 1 mL doses of Ivermectin, two wks

apart (Ivomec; Merial Ltd., Iselin, NJ). To prevent coccidiosis, an amprolium solution

was given as an oral drench, lambs received 1 mL daily in a six d sequence (Corid 9.6%;

Merial, Duluth, GA). On d 21 the animals were dewormed orally with 5cc of

Fenbendazole, (Panacur; Pfizer Animal Health, Exton, PA) and again drenched with 1

mL of Corid from d 21 to 26 (Corid 9.6%).

The lambs were housed (seven to each pen), in covered, earth-floored wooden pens

(24 sq. m), bedded with pine wood chips with adequate bunk space and ad libitum water

and common salt. The University of Florida Institutional Animal Care and Use

committee approved the experimental protocol (D231) used in this study.









A corn-SBM basal diet was formulated to meet NRC (1985) requirements for CP,

TDN, vitamin, and minerals for lambs of this weight and age (Table 3-1). Prior to the

experiment, during a three wk adjustment period, lambs were fed the basal diet at 1200 to

1300 g/d per animal. During the experiment, the animals were fed once daily, 1300

to1600 g-lamb-d-1.

Lambs were stratified by sex and randomly assigned to six dietary treatments; 1)

control (10% sand), 2) (9.7% sand and 0.3% A1C13), 3) (2.5% WTR and 7.5% sand), 4)

(5% WTR and 5% sand), 5) (10% WTR and 0% sand), and 6) (10% WTR, 0% sand, plus

double the quantities of the mineral-vitamin premix, and 1.29% dicalcium phosphate).

The WTR used contained 7.8% total Al on a DM basis. Ten percent of each diet was

either sand, WTR, AlC13 or a combination of the three. The diet concentrations of Al

were 910, 2000, 2000, 4000, 8000, and 8000 ppm, (DM basis) respectively, for the six

diets.

On d 91, animals were placed into individual metabolic crates (1.4m2) to determine

apparent digestibility of P. During a subsequent 21 d crate confinement, all animals were

individually fed their respective experimental diets. Fresh feed was given ad libitum each

morning. Orts were weighed back daily. Individual feed intake, ADG, and BW

differences from wk 11 to wk 14, were evaluated.

Sample Collection, Preparation, and Analyses

Blood samples (jugular venipuncture) and lamb weights were collected on d 0 and

every 14 d thereafter. Blood was collected (10mL) with a 20 x 1 vacutainer (Vacutainer;

Becton Dickinson, Franklin Lakes, NJ) needle into evacuated tubes containing sodium

heparin. Immediately after collection, blood was centrifuged at 700 x g for 30 min, and

plasma was collected and frozen at 0 oC. After a 30 min thaw period, to allow plasma to









reach ambient temperature the proteins were separated using 10% trichoroacetic acid

(Miles et al, 2001).

On d 91, wether lambs were fitted with cloth fecal collection devices for the study

of apparent digestibility of P. Feces were collected daily for 14 d and composite samples

were frozen at 0 C. Each composite sample was sub-sampled and ground in a blender

with stainless steel blades. Feces were then dried for 16 h at 1050C to determine DM.

Samples were then ashed in a muffle furnace at 6000C for 8 h, digested in HC1, filtered,

and diluted for colorimetric P determination (Harris and Popat, 1954).

On d 111, all animals were sacrificed at a USDA approved facility. The following

tissues were collected and analyzed for Al, Ca, Cu, Fe, Mg, Mn, P, and Zn contents:

blood plasma, liver, heart, kidney, and brain and Se was analyzed for the kidney. Samples

were dried, weighed, ashed, and solubilized in HNO3 acid (Miles et al., 2001). Bone was

analyzed for P, Ca, and Mg. For all samples, P was analyzed using a colorimetric

procedure (Harris and Popat, 1954). Kidney Se was determined using fluorometric

procedures (Whetter and Ullrey, 1978). Calcium, Fe, Mg, Cu, Mn, and Zn in tissues and

feed samples were analyzed by flame atomic absorption spectrophotometry (Perkin-

Elmer Model 5000, Perkin-Elmer Corp., Norwalk, CT). Aluminum concentrations were

analyzed in diets, heart, brain, liver, and kidney by atomic absorption spectrophotometer

using nitrous oxide-acetylene flame (Varian SpectrAA 220 FS; Varian Inc., Walnut

Creek, CA).

Statistical Analysis

Soft tissue, fecal, and feed intake data were analyzed for treatment effects using

PROC GLM in SAS (SAS for Windows v9; SAS Inst., Inc. Cary, NC) in a completely

randomized design. PROC MIXED of SAS was used to analyze treatment effects on









BW, ADG, and plasma P as repeated measures with a variance component covariance

structure in respect to d and subplot of animal nested within treatment. Significance was

declared at P < 0.05 and tendencies were discussed when P < 0.15.

Results

Six animals died during the experiment. The cause of death was determined to be

parasite infestation of the gastrointestinal tract, and was deemed unrelated to dietary

treatment. Body weights increased for all treatments for wks 0 to 14 (Table 3-2).

Average daily gains (Table A-i) and feed intakes (Table 3-3) also increased with time (P

< 0.05). Throughout the experiment, lambs fed 2,000 ppm Al via AlC13 consistently had

numerically lower BW than all other treatments. During wk 6, lambs fed 2,000 ppm Al

via AlC13 had lower BW than control animals and, lambs fed 2,000 ppm Al, 4,000 ppm

Al or 8,000 ppm Al from WTR (P < 0.05). Lambs receiving 2,000 ppm, 4,000 ppm and

8,000 ppm Al via WTR were heavier than animals consuming 2,000 ppm Al via AlC13

during wk 11 (P < 0.05). Body weights during wk 11 differed by 11.3 kg, (P < 0.05)

between those animals consuming 2,000 ppm Al via AlC13 and those fed 8,000 ppm Al

via WTR, whereas the difference between these two groups at wk 14 was 7.4 kg (P=

0.008).

During wk 2, ADG of lambs given 8,000 ppm Al from WTR exceeded animals

given 2,000 ppm Al via AlC13 (P < 0.05). Lambs receiving 4,000 ppm Al from WTR

tended (P = 0.11) to gain more than lambs fed 2,000 ppm Al via AlC13. During wk 4

lambs receiving the control, 2,000 ppm Al via WTR and 8,000 ppm Al via WTR

treatments had higher gains than lambs in the treatment given 2,000 ppm Al via AlC13 (P

< 0.05). During wk 6, lambs consuming the control, and 4,000 ppm Al from WTR diets

gained more than lambs consuming 2,000 ppm Al from AlC13 (P < 0.05). Additionally,









during wk 6, all treatments, except 2,000 ppm and 4,000 ppm Al via WTR had gains

much lower than the control (P < 0.05).

During wk 11, animals began a 3-wk individual feeding regime to determine feed

intake. From wk 11 to wk 14, lambs fed the control, 2,000 ppm, and 4,000 ppm Al via

WTR (P < 0.05) consumed more than those fed 8,000 ppm Al via WTR.

Observations of plasma P during wk 4 (Table 3-4) showed that animals receiving

2,000 ppm Al via WTR had higher concentrations than all other treatments, except those

receiving 8,000 ppm Al via WTR plus double the minerals and vitamins (P < 0.05). The

animals receiving 8,000 ppm Al via WTR had lowest plasma P of all groups of animals

(P < 0.05). In wks 6 to 11, the lambs receiving 2,000 ppm Al via AlC13 had lower plasma

P than controls (P < 0.05). During wk 11, both the control and lambs receiving 2,000

ppm Al via WTR had higher plasma P than animals receiving 2,000 ppm Al via AlC13 (P

< 0.05). Analyses of plasma P during wk 14 showed that controls had higher P

concentrations than lambs receiving 4,000 ppm Al via WTR, 8,000 ppm Al via WTR or

8,000 ppm Al via WTR plus two times the amount of added mineral-vitamin premix, and

1.29% dicalcium phosphate (P < 0.05). Plasma evaluations of all other minerals showed,

no differences among treatments, which included the following (.g/ml): Ca 87 to 101,

Mg 17 to 21, Cu 1.3 to 1.5, Fe 0.9 to 2.0, Mn 0.05 to 0.06, and Zn 0.3 to 1.6.

Tissue mineral concentrations (Table 3-5) among treatments were deemed not to be

hazardous to animal health. With the exception of Cu, tissue mineral concentrations

remained within normal ranges (Miles et al., 2001). Liver Cu concentrations were high

for all treatments. The mineral-vitamin premix used, inadvertently contained excess Cu in

relation to sheep requirements. Levels of P showed no differences among treatments









except that animals given 4,000 ppm Al from WTR deposited more P in the kidney than

those animals receiving 8,000 ppm Al from WTR (P < 0.05). No differences (P < 0.05)

were observed in soft tissue or bone Ca concentrations. Aluminum was deposited in

lower amounts in the brain for lambs fed 2,000 ppm Al via WTR than all other treatments

except the control (P < 0.05). Kidney Al deposits were higher in lambs receiving 2,000

ppm Al via AlC13 than those receiving 8,000 ppm Al via WTR (P < 0.05), and those

receiving 8,000 ppm Al via WTR plus two times the added amount of mineral-vitamin

premix, and 1.29% dicalcium phosphate (P < 0.05). Concentrations of Mg showed no

differences in soft tissue deposition. Differences in Fe deposition were observed in liver

(P < 0.05), with lambs consuming the AlC13 treatment having lower Fe concentrations

than those receiving the two treatments of 8,000 ppm Al as WTR. Variations in heart and

kidney Mn concentrations seemed unrelated to Al source or quantity.

Apparent P absorption ranged from -12.9 to 31.8 % (Figure 3-1). The control and

all WTR treatment lambs had a greater apparent P absorption (10.9-31.89%) than the

negative absorption (-12.9%) of lambs fed 2,000 ppm Al via AlC13 (P < 0.001).

Discussion

Increases in BW, ADG and intakes were observed for all treatments and can be

likely attributed to increased appetite which occurs in growing animals. The previous

studies at the University of Florida conducted by Valdivia et al. (1978; 1982) observed an

increase in feed intake from 1.03 to 1.20 g/d, and an increase in BW gain as dietary P was

increased from 0.15 to 0.29 % in diets that contained 1,200 ppm to 2,000 ppm Al as

A1C13. Valdivia et al. (1978) and Rosa et al. (1982) concluded that the increase in P was

able to overcome the clinical signs normally observed with Al toxicosis. Diets in the

present study contained approximately 0.25% P as fed (Table 3-1), which exceeds the









requirements (0.23% dietary P) of lambs of this age and breed (NRC, 1985; McDowell,

2003). Our study showed no major losses in weight or intakes regardless of treatment,

which seems to be attributed to the proper amounts of dietary P (0.25%) supplied. This

concurs with the work of Valdivia et al. (1978) and Rosa et al. (1982).

The control lambs, which received 910 ppm Al from sand, and lambs receiving

treatments containing WTR had no declines in intake. This is likely attributed to the low

bioavailability of Al in WTR and sand (O'Connor et al., 2002; Dayton et al., 2003). A

low bioavailable Al source is much less likely to depress intake because the Al would not

readily react with the P in the gastrointestinal tract.

Aluminum from AlC13 is an available source and has been shown to depress

intakes. Declines in intakes caused by ingestions of an available Al source have been

observed in various species including: sheep (Valdivia et al., 1978; Rosa et al., 1982),

broilers and chicks (Fethiere et al., 1990), humans (Chappard et al., 2003; Rengel, 2004)

and rats (Gomez- Alonso et al., 1996). An Al toxicity results in a P deficiency

(McDowell, 2003) which can lead to serious tissue damage, lower intakes and gains.

Williams et al. (1990; 1991a,c; 1992) induced a P deficiency in heifers and observed an

11% decrease in feed intake. In the present study, there was a decrease in feed intake for

the lambs that were fed 2,000 ppm Al via AlC13. This is expected, as AlC13 is considered

to be a bioavailable source of Al (Valdivia et al., 1978; Rosa et al., 1982), and thus may

induce a P deficiency and depress feed intake.

Ingestion of Al as AlC13 by ruminants decreases bone density, plasma P levels, feed

intakes and gains (Rosa et al., 1982; Valdivia et al., 1982; Ammerman et al., 1984).

Animals receiving the AlC13 diet repeatedly had lower BW and feed intakes than animals









fed other sources of Al. Lower intakes and gains can be attributed to Al availability,

similar observations occurred when 0.75% aluminosilcate was fed to laying hens and

feed intake was significantly depressed (Fethiere et al., 1990).

One of the objectives of the present study was to compare the availability of Al in

WTR to Al in AlC13 and a control when fed to ruminants. During wk 11, body weights

ranged from 36.8 kg for lambs fed 2,000 ppm Al via AlCl3 diet to 48.1 kg for lambs fed

8,000 ppm Al via WTR. Thus, lambs receiving 8,000 ppm Al from WTR, on average,

had BW that were 11.2 kg heavier than those fed 2,000 ppm Al from AlC13 despite the

four fold difference in total Al administered. The group fed 8,000 ppm Al from WTR had

the highest amount of Al and the largest percentage of WTR (10% of the diet as fed).

Differences observed in BW, between lambs fed 8,000 ppm Al via WTR and 2,000 ppm

Al via AlCl3 validates previous studies which showed Al in Al-WTR to be high in a non-

available source of Al (O'Connor et al., 2002; Novak and Watts, 2004) and that AlC13 is

available for uptake in the small intestine (Valdiva et al., 1978; Rosa et al., 1982). It is

thought that grazing ruminants can consume up to 10-15% of their total DM intake as soil

(Healy 1967; 1968). It has also been shown that soil Al is often consumed by grazing

ruminants in amounts as high as 10% of the soil consumed. Aluminum ingested from

soil sources has not been shown to reduce performance. Ammerman et al. (1984) fed

sheep varying soils types, from Latin America, containing as much as 16,600 ppm Al.

They concluded that the soil Al sources had no significant effect on BW, gains, and

intakes of the sheep which consumed them. The soils contained various levels of Al or

Fe oxides, which is similar to the chemical form of Al from WTR. The additions of high

Fe and Al soils had no harmful effects on P utilization, feed intake, or gains.









Differences, in general, between treatments were limited throughout the trial.

Lambs receiving diets containing Al via WTR at varying levels showed no differences in

BW from the control (P < 0.05). Additions of WTR in amounts as high as 10% of the

diet, and representing 8,000 ppm Al in the diet, do not negatively impact growing lambs

in relation to BW, ADG, and feed intakes when dietary P is at least 0.25%. Thus, under

natural grazing conditions, [where 10% of the DM intake is of soil (Field and Purves,

1964; Healy 1967; 1968)], even high rates of surface applied WTR are not expected to

harm animal performance.

During wk 14, the ADG of treatments plateaued, consistent with a natural

sigmoidal growth curve. Prior to wk 14, animals were gaining at rates between 463 to

593 g per d. The rate declined during wk 14 to only 207 to 244 g per d, but the decline is

not attributed to dietary treatments. Animals appeared healthy with notable

accumulations of body fat. Lambs in both the control and AlC13 treatment continued to

gain larger amounts of weight during wk 14, because they had not reached a maintenance

weight. Lambs fed 2,000 ppm Al via AlC13 had lowered growth, intake and BW

throughout the trial and had not reached a growth plateau by wk 14. The control animals

during wk 6 experienced an illness which was attributed to parasite infestations which

suppressed ADG means thereafter. In previous studies, similar declines in ADG were

observed with AlC13 additions, and animal growth peaked at later dates than those not

receiving an Al source (Valdivia et al., 1978; Rosa et al., 1982; Fethiere et al., 1990).

Intakes, regardless of treatment, increased with time. Constituents added to the

basal diets did not cause any animals to become anorexic, a common clinical sign of Al

toxicity, or P deficiency (Williams et al., 1992; McDowell, 2003). Differences in intakes









were evaluated individually in a 3-wk period between wk 11 to 14. Prior to this date,

lambs had been group fed. Individual intake data were similar to those reported by Rosa

et al. (1982), and Valdivia et al. (1978). Lambs fed diets containing 2,000 ppm Al from

A1C13 consumed less than the control (P > 0.05), which can again be attributed to the high

bioavailability of AlC13. Intakes were the lowest for animals consuming 8,000 ppm Al

from WTR. During wk 14, these animals had the highest BW, but a decline in ADG

from wk 11 (480 g) to wk 14 (207.0 g), which was the lowest gain for that period.

Intakes for lambs receiving 8,000 ppm Al from WTR were lower than the control, 2,000

ppm and 4,000 ppm Al from WTR (P < 0.05), but higher than lambs receiving 2,000 ppm

from AlC13 or 8,000 ppm Al from WTR with additional minerals and vitamins, (P >

0.05). Prior to wk 14, lambs fed 8,000 ppm Al from WTR showed adequate performance

in relation to gains, intake and BW. Therefore, the cause of these declines seen in lambs

receiving 8,000 ppm Al via WTR are unknown and could be related to normal growing

patterns, an unknown parasite infestation, Cu toxicities, Al toxicities, or other various

environmental interactions.

During wk 4, lambs receiving 2,000 Al from WTR had the highest concentration of

plasma P and differed from the control, those receiving 2,000 ppm Al from AlC13, 8,000

ppm Al from WTR. (P < 0.05). Huff et al. (1996) administered 3.7% aluminum sulfide

to broiler chicks and observed a declines in serum P after a 3 wk period. Lambs in the

present study, had plasma P levels decline from 54.2 [g/ml to 19.6 [g/ml, between wk 4

and wk 8. Additionally, all treatments showed declines in plasma P during this period,

but the AlC13 treatment declines were most often the greatest. During wk 11 and 14,

plasma P concentrations began to increase in all treatments. One could conclude that









plasma P concentrations declined to levels which demanded the use of body stores of P

(Williams et al., 1990; McDowell, 2003). Bone mineral content was evaluated in the

long bones, with no differences among treatments and no evidence of a mineral

depression; yet research has shown that the ribs and the vertebrae are first to become

depleted in mineral concentrations (Williams et al., 1990; 1991a; McDowell, 2003).

Therefore the possibility exists that increases in plasma P levels during wk 11 to 14

occurred from bone mineral resorption. This is unlikely, but not unreasonable, because

within a long time frame of 8 wk (between wk 6 and wk 14) bone loss most likely would

have been observed in the long bone of the leg which was analyzed. Previous

experimental data have not demonstrated similar results by showing an increase in

plasma P after a decline. Therefore, observations are speculative at this time and further

research is needed to validate this theory.

Tissue mineral concentrations analyzed for this study were in the normal ranges for

lambs of this breed and age (Miles et al., 2001; McDowell 2003). Previous research

found differences in kidney, bone, liver and spleen concentrations of Al, Fe, P Mg and Zn

(Rosa et al., 1982) and Ca (Rosa et al., 1982; Zafar et al., 2004) when various amounts of

Al were fed. In the present study, Al concentrations differed in brain, heart, liver, and

kidney, Mg in bone, and Fe in the liver. Absorption of Al in mongastrics is

approximately 0.1% (Rengel, 2004) and is thought to be even lower in ruminants

(Valdivia et al., 1970; 1978). Aluminum accumulation occurs most readily in the brain.

The exact mechanism is unknown but Al can cross the blood-brain barrier (Rengel,

2004). Accumulations of Al in brain tissue were greater from lambs fed 2,000 ppm Al

from AlC13, than from lambs fed 2,000 ppm from WTR (P < 0.05). Aluminum









concentrations in brains increased when Al from WTR was fed at levels higher than

2,000 ppm, but did not differ from the control. Liver depositions of Al were highest in

lambs fed 8,000 ppm Al from WTR (P < 0.05). In the kidney, the highest concentrations

of Al were detected when lambs were fed 2,000 ppm Al from AlC13, and differed from

both treatments receiving 8,000 ppm Al from WTR and from the control and 2,000 ppm

Al via WTR (P < 0.05). Rosa et al. (1982) observed increases in Al tissue concentration

as Al consumption increased, which was not consistently observed in our study.

Additionally, soft tissues, except brain matter, that have been evaluated in past studies

have not been shown to accumulate large amounts of Al during short time periods

(Rengel, 2004), and may not prove to be useful for determination of differences of any Al

sources and levels.

Apparent P absorption from a 14 d fecal collection showed differences among all

five treatments versus the treatment containing 2,000 Al via AlC13. Studies by Valdivia

et al. (1982) observed a marked decrease in P absorption and net P retention in lambs fed

2,000 ppm Al as AlC13. Negative apparent P absorptions were observed in all groups

except those given high P with low Al. When 0.29% P was fed with no dietary Al, the

mean apparent absorption was unaffected. In our study, the control had an apparent

absorption of 22.5%, and the mean for all the WTR groups was 21.2%. This suggests

that Al in WTR did not negatively impact or reduce dietary P absorption. Valdivia et al.

(1982) found a negative apparent P absorption (-10.7%) when 0.29% dietary P and 2,000

ppm Al as AlC13 were fed to sheep. Additionally, Martin et al. (1969) conducted P

retention studies using dietary applications of a hydrated Al source and discovered that

when Al was fed to sheep, retained amounts of P decreased linearly, as Al fed increased









from 910, 2000, 4000, 8000, and 8000 ppm. Similar results were observed in our study

when 2,000 ppm Al was added via AlC13 to the basal diet which contained 0.25% P. The

apparent P absorption averaged 12.9% at wk 14 and therefore suggested a negative

impact on dietary P utilization with added Al as AlC13.

Summary and Conclusions

A 14 wk experiment was conducted using 36 lambs. Individual feeding was

recorded between wk 11 to 14. Diets, containing 0.25% P (as fed), included 1) control

(10% sand), 2) (9.7% sand and 0.3% AlC13), 3) (2.5% WTR and 7.5% sand), 4) (5%

WTR and 5% sand), 5) (10% WTR and 0% sand), and 6) (10% WTR, 0% sand, double

the added quantities of the mineral-vitamin premix, and 1.29% dicalcium phosphate).

The Al varied from 910 to 8,000 ppm among diets. Lambs fed the control and WTR had

no decline in intake, but the AlC13 lambs repeatedly had lower BW and intakes. The

WTR contain a non-available source of Al and did not cause performance declines

Additions of this WTR respecting Al concentrations as high as 8,000 ppm, did not

negatively impact growing ruminants in relations to BW, ADG, and intakes. During wk

6, all treatments showed declines in plasma P, but the AlC13 treatment was often the

lowest, and during wk 11 plasma P began to increase.

Accumulations of Al in the brain were greatest for lambs given 2,000 ppm Al from

A1C13 and increased numerically when Al as WTR was fed at levels higher than 2,000

ppm. With the exception of the brain, soft tissues did not accumulate large amounts of Al

during this 14 wk experiment.

Apparent P absorption from a 14 d metabolic study was positive (10.9-31.8%) for

all lambs fed the control and various levels of WTR. However, lambs that received 2,000

ppm Al via AlC13 had a negative P absorption of -12.9 %. This was a lowered (P < 0.03)









P absorption compared to all other treatments. Aluminum, as AlC13, fed at 2,000 ppm

reduced dietary P retention, but varying amounts of Al as WTR had no effect on P

apparent absorption with similar absorption rates as the control. Therefore when dietary

P is supplied in amounts of 0.25% or higher, Al (as WTR) fed to lambs in amounts as

high as 8,000 ppm did not negatively impact the feed intake, gain, BW or P absorption.

Implications

Dietary administration of AlC13 has negative impacts on ADG, BW, feed intake,

apparent absorption of P, and P plasma concentrations. Lambs fed WTR had apparent P

absorption percentages that were similar to the control and were higher than the AlC13

treatment. Water treatment residuals are not harmful when consumed in amounts up to

8,000 ppm Al, when P is supplied in amounts of 0.25%, and do not negatively affect

gain, feed intake, BW, or P availability.










Table 3-1. Diet composition (as-fed) and analyses for average (n=18) concentrations
for macro- and micro-elements for treatments
Treatments


Ingredient (%,as fed)
Ground Corn
Soybean hulls
Wet molasses, unfortified
Cottonseed hulls
Corn gluten meal, 60% CP
Alfalfa meal, 17% CP
Vegetable oil (soybean)
Sandb
Water treatment residual
Aluminum chloride
Salt
Urea
Ground limestone
Ammonium chloride
Flowers of sulfur
Mineral-Vitamin-premixd
Dicalcium phosphate

Analyses (ppme)
Ca
Mg
Na
K
P
Al
Co
Cu
Fe
Mn
Zn


1
41.1
12.5
10.0
8.0
5.5
5.0
4.0
10.0


1.0
1.6
0.7
0.5
0.01
0.01



7,170
2,780
4,060
4,180
2,520
910
7
31
66
11
74


2
41.1
12.5
10.0
8.0
5.5
5.0
4.0
9.3

0.7
1.0
1.6
0.7
0.5
0.01
0.01



7,120
2,730
3,240
5,210
2,490
2,320
5
33
65
13
70


3
41.1
12.5
10.0
8.0
5.5
5.0
4.0
7.5
2.5

1.0
1.6
0.7
0.5
0.01
0.01



7,220
2,700
3,030
3,960
2,550
2,270
6
33
67
13
71


4
41.1
12.5
10.0
8.0
5.5
5.0
4.0
5.0
5.0

1.0
1.6
0.7
0.5
0.01
0.01



7,440
2,880
3,000
4,560
2,480
3,970
5
32
66
13
70


5 6
41.1 39.9
12.5 12.5
10.0 10.0
8.0 8.0
5.5 5.5
5.0 5.0
4.0 4.0

10.0 10.0

1. 1.0
1.6 1.6
0.7 0.7
0.5 0.5
0.01 0.01
0.01 0.02
1.3


7,300
2,870
3,410
4,440
2,460
7,860
5
34
66
14
67


10,000
3,020
3,110
4,380
5,020
7,790
8
42
70
19
79


aDietary treatments were created by additions to a corn-SBM basal diet as follows: 1) (Control) 10% sand; 2)
9.3% sand + 0.7% AlC13; 3) 7.5% sand, + 2.5% WTR; 4) 5% sand + 5%WTR; 5) 10% WTR; 6) 10% WTR +
two times the added amount of mineral-vitamin premix + 1.29% dicalcium phosphate; Treatments 2 and 3
were formulated to contain 2,000 ppm Al, treatment 4 was formulated to contain 4,000 ppm Al, and
treatments 5 and 6 were formulated to contain 8,000 ppm Al.
b Sand contained 0.2 % Fe, 0.01 % Al, 0.09% Ca, 0.03 % Mg, 0.1 % P, 0.005 % Mn, 0.004 % Cu, and 0.001
% Zn.
' Water Treatment Residuals contained 0.30 % Fe, 7.8 % Al, 0.11 % Ca, 0.024 % Mg, 0.3 % P, 0.004 % Mn,
0.73 % S, 0.006 % Cu, and 0.002 % Zn.
dNlineral-Vitamin-premix contained 8.0% Mg (as oxide), 0.70% Fe (as sulfate), 2.40% S (as sulfate), 1.9 %
Cu (as sulfate), 6.0 % Mn (as oxide), 0.47 % I (as iodate), 0.075 % Se (as sodium selenite), 4.5 % Zn (as
oxide and sulfate), 133,363.4 IU/kg Vitamin A supplement, 412,272.7 IU/kg Vitamin D3 supplement, 259.1
IU/kg Vitamin E supplement, rice mill byproduct, and stabilized fat as a vitamin carrier.
eDry matter basis.









Table 3-2. Effects of dietary Al concentration and source on BW of feeder lambsa
Treatment
1 2 3 4 5 6 SE
BW, kg
Wk
0 32.6 31.4 33.1 31.1 32.2 31.7 1.5
2 32.8cd 31.6c 34.6cd 34.4cd 37.5d 33.6cd 1.9
4 34.3cd 31.6c 37.7de 35.8cde 41.3e 35.2cd 2.3
6 38.4d 32.3c 40.7d 39.3d 41.2d 34.7cd 2.6
11 41.4cd 36.8c 46.7d 45.1d 48.1d 42.9cd 2.5
14 49.3cd 45.9c 52.8d 50.3d 53.3d 49.7cd 1.9
aData represent least squares means; n = 7 per treatment
bDietary treatments were created by additions to a corn-SBM basal diet as follows: 1)
(Control) 10% sand; 2) 9.3% sand + 0.7% AlC13; 3) 7.5% sand, + 2.5% WTR; 4) 5% sand
+ 5%WTR; 5) 10% WTR; 6) 10% WTR + two times the added amount of mineral-vitamin
premix + 1.29% dicalcium phosphate; Treatments 2 and 3 were formulated to contain 2,000
ppm Al, treatment 4 was formulated to contain 4,000 ppm Al, and treatments 5 and 6 were
formulated to contain 8,000 ppm Al.
"deMeans within rows lacking a common superscript differ (P < 0.05).








Table 3-3. Effect of dietary Al concentration and source on feed intake of feeder lambsa
Treatment
1 2 3 4 5 6 SE
intake, g-lamb1-ld-1
Wk
2 827 959 1170 1120 1100 1020
4 1410 876 1150 1150 1070 1120
6 954 1110 1150 1200 1210 1210
11 1610 1460 1790 1550 1910 1940
14 1940c 1870cd 1900c 1940c 1270d 1570cd 54.6
aData represent means of intake during wk 0 to 11 and least squares means during wk 14;
n = 7 per treatment
bDietary treatments were created by additions to a corn-SBM basal diet as follows: 1)
(Control) 10% sand; 2) 9.3% sand + 0.7% AlC13; 3) 7.5% sand, + 2.5% WTR; 4) 5% sand
+ 5%WTR; 5) 10% WTR; 6) 10% WTR + two times the added amount of mineral-vitamin
premix + 1.29% dicalcium phosphate; Treatments 2 and 3 were formulated to contain 2,000
ppm Al, treatment 4 was formulated to contain 4,000 ppm Al, and treatments 5 and 6 were
formulated to contain 8,000 ppm Al.
cd Lambs were individually fed for 3 wk ending at wkl4; Means within rows lacking a
common superscript differ (P < 0.05).









Table 3-4. Effect of dietary Al concentration and source on plasma P of feeder lambsa
Treatment
1 2 3 4 5 6 SE
--g/ml,
Wk
0 48.7 44.6 51.2 40.8 45.6 43.8 3.7
2 48.3 44.7 50.5 41.5 45.7 44.1 5.3
4 54.3d 54.2d 64.4c 49.8d 39.7e 58.5cd 4.6
6 39.2c 25.2d 27.9d 28.5d 26.2d 27.0d 2.5
8 33.8c 19.6d 19.9d 26.3cd 21.9d 28.1cd 2.3
11 36.2cd 22.2e 46.3c 30.0de 29.0de 29.3de 5.0
14 38.3c 34.0cd 34.9cd 28.0de 24.5de 24.9e 3.6
aData represent least squares means; n = 7 per treatment
bDietary treatments were created by additions to a corn-SBM basal diet as follows: 1)
(Control) 10% sand; 2) 9.3% sand + 0.7% AlC13; 3) 7.5% sand, + 2.5% WTR; 4) 5% sand
+ 5%WTR; 5) 10% WTR; 6) 10% WTR + two times the added amount of mineral-vitamin
premix + 1.29% dicalcium phosphate; Treatments 2 and 3 were formulated to contain 2,000
ppm Al, treatment 4 was formulated to contain 4,000 ppm Al, and treatments 5 and 6 were
formulated to contain 8,000 ppm Al.
"deMeans within rows lacking a common superscript differ (P < 0.05).











Table 3-5. Tissue mineral composition resulting from experimental diets
Treatment


-Macro elements, (o%)


0.01
0.08
0.04
38.1

0.15
0.11
0.066
0.79c


0.01
0.08
0.05
39.5

0.14
0.13
0.070
0.79c


1.16 1.16
1.21cd 1.17
1.07 1.09
14.6 15.4
-Micro elements, (mg/kg)


Ca
Heart
Kidney
Liver
Bone
Mg
Heart
Kidney
Liver
Bone


0.01
0.06
0.05
39.9

0.15
0.11
0.066
0.79c

1.16
1.12c
1.07
14.2


43.1cd
6.9cd
7.2cd
15.4

8.7
38.0
4,090

152
443
212cd

1.6cd
18.3cd
12.8

1.1


0.01
0.04
0.04
39.4

0.13
0.11
0.062
0.70cd

1.03
1.15cd
1.17
14.4


52.0d
5.1 def
9.4
22.3cd

12.7
37.3
4,570

143
442
141d

1.4d
23.2cd
11.4

1.2


0.01
0.06
0.04
37.0

0.12
0.10
0.062
0.73cd

1.09
1.15 d
1.05
14.1


47.5d
7.0cd
5.4d
25.3d

10.3
31.2
3,270

162
434
262c

1.57cd
23.9cd
11.9

1.1


Heart
Kidney
Liver


71.5
83.9
48.5


63.6
110
48.4


65.7
120
44.8


69.1
110
43.5


59.8
85.1
36.8


6.65
15.0
6.91


aData represent least squares means; n = 5, 5, 7, 7, 5, and 6 for the control and treatments 1-
5, respectively
bDietary treatments were created by additions to a corn-SBM basal diet as follows: 1)
(Control) 10% sand; 2) 9.3% sand + 0.7% AlC13; 3) 7.5% sand, + 2.5% WTR; 4) 5% sand
+ 5%WTR; 5) 10% WTR; 6) 10% WTR + two times the added amount of mineral-vitamin
premix + 1.29% dicalcium phosphate; Treatments 2 and 3 were formulated to contain 2,000
ppm Al, treatment 4 was formulated to contain 4,000 ppm Al, and treatments 5 and 6 were
formulated to contain 8,000 ppm Al.
"defMeans within rows lacking a common superscript differ (P < 0.05).


50.4 d
7.4
7.5d
20.9cd

14.4
36.1
3,930

146
447
162cd

1.31d
23.2d
10.2

1.1


Heart
Kidney
Liver
Bone


Al
Brain
Heart
Kidney
Liver
Cu
Heart
Kidney
Liver
Fe
Heart
Kidney
Liver
Mn
Heart
Kidney
Liver
Se
Kidney


33.9
6.1 cdef
7.1cd
16.7

9.8
46.9
3,080

154
425
208cd

1.80C
16.4c
14.1

1.3


1.2 0.07


0.02
0.05
0.05
37.7

0.13
0.10
0.064
0.61d

1.01
1.14cd
1.01
15.1


48.2d
4.5ef
5.4d
18.5cd

7.4
27.9
3,900

134
434
226c

1.50cd
18.4cd
11.4


0.004
0.02
0.01
18.3

.019
0.02
0.004
0.04

0.14
0.087
0.18
0.63


4.10
0.79
1.03
2.75

2.07
9.79
713

10.9
66.4
27.8

0.13
2.4
2.0










40.0


31.8


b
22.5








12.9

-12.9


18.2


24.0 b

b
10.9



5 6


Treatments

Figure 3-1 Effect of dietary Al source on apparent P absorption. Dietary treatments were
created by additions to a corn-SBM basal diet as follows: 1) (Control) 10% sand; 2) 9.3%
sand + 0.7% AlC13; 3) 7.5% sand, + 2.5% WTR; 4) 5% sand + 5%WTR; 5) 10% WTR;
6) 10% WTR + two times the added amount of mineral-vitamin premix + 1.29%
dicalcium phosphate; Treatments 2 and 3 were formulated to contain 2,000 ppm Al,
treatment 4 was formulated to contain 4,000 ppm Al, and treatments 5 and 6 were
formulated to contain 8,000 ppm Al. The SE for treatments is 8.23.
aMeans lacking a common superscript differ (P < 0.05).


30.0

20.0

10.0

0.0

-10.0


-20.0














CHAPTER 4
EFFECT OF DIETARY ALUMINUM FROM WATER TREATMENT RESIDUALS
ON BONE DENSITY AND BONE MINERAL CONTENT OF FEEDER LAMBS

Introduction

Animal waste, which is often applied to grazing land, contains P that can remain in

the A horizon of the soil profile and may lead to water pollution (Haustein et al., 2000).

Aluminum (Al), if applied to the land, is thought to complex with P and to reduce the

soluble P concentrations in animal waste (O'Connor et al., 2002). This type of reduction

in soluble P levels of animal waste products could result in significant decreases in

pollution by commercial livestock operations.

Previous studies suggest that the application of Al containing water treatment

residuals (WTR), a byproduct of water purification, increases the soil's capacity to bind

and retain P (Elliott et al., 2002). The chemical mixture of soil and WTR has been shown

to increase the retention value of soil P by several fold (Novak and Watts, 2004) and

could result in a possible solution to environmental concerns associated with animal

waste disposal. A major concern is that WTR, containing high amounts of Al, may

adversely affect P utilization and bone deposition in grazing livestock that inadvertently

consume WTR.

Highly available dietary Al may create unabsorbable P complexes in the intestinal

tract and, Al toxicosis is often observed as a P deficiency (Valdivia, 1977). Diets fed to

sheep containing 0.29% P and 2,000 ppm Al via AlC13 resulted in reduced bone density

(Rosa et al., 1982). Additionally, high amounts ofbioavailable Al can also negatively









impact the status of Fe, Zn, and Mg in sheep (Rosa et al., 1982). The bioavailability of Al

varies in different WTR, but is generally much lower than Al compounds such as AlC13

(O'Connor et al., 2002). The purpose of this study was to determine the effect of dietary

Al as WTR and AlC13 on bone mineral content (BMC) and bone density in feeder lambs.

Materials and Methods

Animals, Diets, and Management

Forty-two, (30 wether and 12 female) five to eight-mo-old lambs, (22 Suffolk and

14 Suffolk-crosses) were utilized in a 14 wk experiment at the University of Florida

Sheep Nutritional Unit located in Gainesville, Florida. The experiment was conducted

from June 6th until September 25, 2004. The lambs weighed between 22 to 39 kg at d

zero Prior to the experiment, lambs received an 8-way Clostridial vaccination given as

injections of 2 mL four wks apart (Ultra Choice 8; Pfizer Animal Health, Exton, PA) and

were dewormed, with two 1 mL doses with Ivermectin, two wks apart (Ivomec; Merial

Ltd., Iselin, NJ). To prevent coccidiosis, amprolium (Corid 9.6%; Merial, Duluth GA)

was used as an oral drench with lambs receiving 1 mL daily in a five d sequence.

Corn-SBM basal diets were formulated to meet NRC (1985) requirements for CP,

TDN, minerals and vitamins for lambs of this weight and age (Table 4-1). Lambs were

fed the basal diet at 1200 to 1300 g-lamb--d-1 during a 3 wk adjustment period and 1300

to 1600 g-lamb-l-d- throughout the experiment. Lambs were stratified by sex and

randomly assigned to six dietary treatments: 1) control (10% sand), 2) (9.7% sand and

0.3% A1C13), 3) (2.5% WTR and 7.5% sand), 4) (5% WTR and 5% sand), 5) (10% WTR

and 0% sand), and 6) (10% WTR, 0% sand, plus double the quantities of the mineral-

vitamin premix, and 1.29% dicalcium phosphate). The WTR was analyzed to contain

7.8% Al on a DM basis. Ten percent of each diet was either sand, WTR or the









combination of the two. The diet concentration of Al were 910, 2000, 2000, 4000, 8000,

and 8000 ppm respectively for the six diets.

Lambs were housed (seven to each pen) covered, in earth-floored wooden pens (24

sq. m), which were bedded with pine wood chips. Animals were group fed in open

troughs until d 84, when animals were placed in individual raised metabolic crates. All

lambs had access to salt and adlibitum water. Dietary treatments were offered adlibitum

and DM intake was monitored daily. Diets were not reformulated during the study.

Sample Collection and Analyses

Radiographic photometry was used to estimate bone mineral content (BMC) at 28

d, 69 d and 109 d. For each lamb, the left dorsal/palmer, third metacarpal region of the

leg was radiographed with the use of a portable x-ray machine, (Easymatic Super 325;

Universal X-Ray Products, Chicago, IL). The machine was set at 97 pkv, 30 ma, and

0.067 sec. One cm below the nutrient foramen of the third metacarpal, a cross section of

the cannon bone was compared to the standard using the image analyzer and BMC was

estimated by photodensitometry. A ten-step Al wedge, taped to the cassette parallel to

the third metacarpal, was used as a standard in estimating BMC. Radiographs were taken

with a 91.5 cm distance between the x-ray machine and the cassette (Meakim et al., 1981;

Ott et al., 1987). The films were processed with an auto-radiograph processing machine,

with Kodak products, and by Kodak development procedures (Eastman Kodak Co.,

Rochester, NY).

Radiographs were evaluated with a photometer (Photvolt Corp., New York, NY);

percentage light transmissions (%T) were used to determine solid matter. The

radiographs were zeroed using the thinnest Al step that could be distinguished as a









differing shade from the next ascending step. The BMC was evaluated 2 cm descending

from the nutrient foramen. Bone diameter and medullar width were taken to the nearest

0.2 mm using a plastic transparent ruler (Meakim et al., 1981). Determination of the %T

reading was then analyzed graphically using a logarithmic calculation. The width of the

bone segment, 2 cm below the nutrient foramen, was compared to the visible segments of

the Al step wedge.

On d 111, all animals were sacrificed at a USDA inspected facility and bone from

left leg was removed for bone mineral and bone density analysis. To prepare bone for P,

Ca, and Mg analyses, bone removed the left dorsal/palmer, third metacarpal region of the

leg, was skinned, immediately wrapped in 0.9% saline-soaked cheese cloth and frozen at

0 C. After thawing, bone was cut into 2 cm sections, 2 cm below the nutrient foramen,

and marrow was carefully removed. Samples were rinsed in deionized water and blotted

dry. Specific gravity procedures (Kit ME-40290; Mettler Instruments Corp., Hightstown,

NJ) were used to determine bone density (g/cm3) as described by Williams et al. (1990,

1991c).

Bone samples were then dried at 105 oC for 16 h, extracted with an ether soxhlet

apparatus for 48 h, air dried for 10 h, and then oven dried at 1050C again for 16 h. Dry

samples were weighed, and ashed in a muffle furnace at 6000C for 8 h. All P samples

were analyzed with colorimetric procedures (Harris and Popat, 1954), while Ca and Mg

were analyzed by flame atomic absorption spectrophotometry (Perkin-Elmer Model

5000, Perkin-Elmer Corp., Norwalk, CT).









Statistical Analysis

Bone density and BMC data were subjected to the GLM procedure of SAS (SAS

for Windows v9; SAS Inst., Inc. Cary, NC) in a completely randomized arrangement.

Significance was declared at P < 0.05, and tendencies were recognized at P < 0.15.

Results

Radiograph BMC

At d 28, lambs receiving 2,000 ppm Al from WTR tended (P = 0.07) to have

greater bone density than lambs receiving 2,000 ppm Al as AlC13 (Table 4-2). Likewise,

lambs receiving 8,000 ppm Al as WTR tended (P = 0.11) to have denser bone then those

receiving 2,000 ppm Al via AlC13. Overall, bone density was unaffected (P = 0.51) by

treatment at d 69, as only lambs receiving 4,000 ppm Al from WTR had bones which

tended to be more dense than the controls. Near the end of the study, (d 109) bone

density was again unaffected by dietary Al content. Only bones from lambs receiving

8,000 ppm Al as WTR tended to be more dense (P = 0.15) than the controls, and no other

difference or tendencies were observed.

Bone Density via Specific Gravity

Bone density as determined by specific gravity (Williams et al., 1990; 1991) was

unaffected by treatment (P = 0.43). Bone density ranged from 1.88 to 1.94 g/cm3 and

bones from lambs receiving 2,000 ppm Al as WTR tended to be more dense (P = 0.07)

than bones from lambs receiving 4,000 ppm Al from WTR.

Bone Mineral Analyses

Bone mineral percentages (Table 4-3) of P and Ca were unaffected by treatment.

Bone mineral percentages of Ca were also unaffected by treatment (P = 0.87). Bone Mg

content differed (P < 0.05) in the lambs receiving 8,000 ppm from WTR plus double the









minerals and vitamins, having lower bone deposits of Mg than those fed the control,

2,000 ppm Al via WTR, or 4,000 ppm Al via WTR (P < 0.05). Additionally, lambs

receiving 4,000 ppm Al from WTR tended to have higher bone Mg content than lambs

fed 8,000 ppm Al from WTR (P = 0.06).

Discussion

Radiographing techniques and specific gravity measurements revealed that dietary

Al content had no effect on BMC or bone density. Bone density and P deposition is

expected to decline when additional Al is ingested according to research conducted by

Valdiva et al. (1977) and Rosa et al.(1982). Diets formulated with high Al content (up to

2,000 ppm) in previous work with mallard ducks and chicks (Capdevielle et al., 1998)

and rats caused a decline (Gomez- Alonso et al., 1996; Zafar et al., 2004) in bone mineral

declines with Al dietary additions. Yet, contradictory results have been described in

much of the research conducted with ruminant species ( Valdivia et al., 1978; 1982; Rosa

et al., 1982) Valdivia (1982) concluded that ruminants are less susceptible to toxic effects

of Al than in other species. Normally, bone ash is 17.6 % P, and 37.7 % Ca (McDowell,

2003). Studies conducted by Validiva et al. (1982) showed bone mineral percentages of

P that were slightly below average, 14 to 15%, as is seen in our study. However in the

present study, differences were not observed among dietary treatments. Since differences

were not observed, there was no notable effect of dietary Al intake on bone mineral

deposition. The long bones of the appendages are often the last affected by P deficiencies

(Williams et al., 1991a,b,c; McDowell, 2003). This could be a logical justification for

the lack of treatment effect in the present study. A secondary explanation is that the

dietary levels of P (0.25%), as seen in intake studies by Rosa et al. (1982), were high

enough to compensate for the Al additions to the basal diet. Rosa et al. (1982) also









reported that bone ash Ca levels were unaffected by dietary Al and ranged from 35 to 36

%, similar to results in our study which ranged from 33.1 to 39.9 % for bone ash Ca.

Studies conducted with rodents (Cox and Dunn, 2001; Zafar et al., 2004) found that Ca

deposits in the bone declined when dietary levels of Ca were deficient and Al was fed. In

the present study, Ca levels were above the requirement and no declines in Ca bone

deposits were observed. The absence of treatment effect is attributed to proper dietary Ca

levels and Ca to P ratios.

Summary and Conclusions

A 111 d experiment was conducted to determine if the use of Al sources (AlC13 vs.

WTR) at various levels (910 to 8,000 ppm) affected BMC and bone density of growing

sheep. Forty-two, 5 to 8-mo-old lambs, (12 ewe and 30 wethers) were utilized in a

completely randomized experimental arrangement. Treatment, consisted of the following:

1) control (10% sand), 2) (9.7% sand and 0.3% A1C13), 3) (2.5% WTR and 7.5% sand),

4) (5% WTR and 5% sand), 5) (10% WTR and 0% sand), 6) (10% WTR, 0% sand,

double the quantities of the mineral-vitamin premix, and 1.29% dicalcium phosphate).

Basal diets met all requirements and contained 0.25% P. The lambs weighed between 22

to 39 kg at d zero and between 45.9 to 53.3 kg on d 111. The WTR contained 7.8% Al

on a DM basis. Ten percent of each diet was either sand, WTR, AlC13 or the combination

of two. The resulting concentrations of Al were 910, 2000, 4000, and 8000 ppm,

respectively, for the six diets.

On d 28, 69, and 109, radiographs were taken. Mean bone densities from

radiographs were similar among treatments (P = 0.30). At experimental termination, d

111, animals were sacrificed. The third metacarpal was then used for specific gravity

procedures, and no differences were observed among treatments (P > 0.40). Overall,









results indicate that Al in various forms and levels fed to growing sheep provided

adequate amounts of P (0.25%) and other required dietary nutrients had no effect on bone

density over a period of 79 d or on specific gravity calculations of bone density over 111

d.

Implications

When sheep received adequate dietary concentrations of P (0.25%), Al from A1C13

or WTR had no effect on bone density or composition. In relation to bone development,

the Al-WTR that contains 7.8% Al, which was implemented, is safe for consumption by

sheep up to 10% of their total diet.










Table 4-1. Diet composition (as-fed) and analyses for average (n=18) concentrations for
macro- and micro-elements for treatments
Treatments


Ingredient (%,as fed)
Ground Corn
Soybean hulls
Wet molasses, unfortified
Cottonseed hulls
Corn gluten meal, 60% CP
Alfalfa meal, 17% CP
Vegetable oil (soybean)
Sandb
Water treatment residual
Aluminum chloride
Salt
Urea
Ground limestone
Ammonium chloride
Flowers of sulfur
Mineral-Vitamin-premixd
Dicalcium phosphate

Analyses (ppme)
Ca
Mg
Na
K
P
Al
Co
Cu
Fe
Mn
Zn


1
41.1
12.5
10.0
8.0
5.5
5.0
4.0
10.0


1.0
1.6
0.7
0.5
0.01
0.01



7,170
2,780
4,060
4,180
2,520
910
7
31
66
11
74


2
41.1
12.5
10.0
8.0
5.5
5.0
4.0
9.3

0.7
1.0
1.6
0.7
0.5
0.01
0.01



7,120
2,730
3,240
5,210
2,490
2,320
5
33
65
13
70


3
41.1
12.5
10.0
8.0
5.5
5.0
4.0
7.5
2.5

1.0
1.6
0.7
0.5
0.01
0.01



7,220
2,700
3,030
3,960
2,550
2,270
6
33
67
13
71


4
41.1
12.5
10.0
8.0
5.5
5.0
4.0
5.0
5.0

1.0
1.6
0.7
0.5
0.01
0.01



7,440
2,880
3,000
4,560
2,480
3,9710
5
32
66
13
70


5 6
41.1 39.9
12.5 12.5
10.0 10.0
8.0 8.0
5.5 5.5
5.0 5.0
4.0 4.0

10.0 10.0

1. 1.0
1.6 1.6
0.7 0.7
0.5 0.5
0.01 0.01
0.01 0.02
1.3


7,300
2,870
3,410
4,440
2,460
7,860
5
34
66
14
67


10,000
3,020
3,110
4,380
5,020
7,790
8
42
70
19
79


aDietary treatments were created by additions to a corn-SBM basal diet as follows: 1) (Control) 10% sand; 2)
9.3% sand + 0.7% AlC13; 3) 7.5% sand, + 2.5% WTR; 4) 5% sand + 5%WTR; 5) 10% WTR; 6) 10% WTR +
two times the added amount of mineral-vitamin premix + 1.29% dicalcium phosphate; Treatments 2 and 3
were formulated to contain 2,000 ppm Al, treatment 4 was formulated to contain 4,000 ppm Al, and
treatments 5 and 6 were formulated to contain 8,000 ppm Al.
b Sand contained 0.2 % Fe, 0.01 % Al, 0.09% Ca, 0.03 % Mg, 0.1 % P, 0.005 % Mn, 0.004 % Cu, and 0.001
% Zn.
' Water Treatment Residuals contained 0.30 % Fe, 7.8 % Al, 0.11 % Ca, 0.024 % Mg, 0.3 % P, 0.004 % Mn,
0.73 % S, 0.006 % Cu, and 0.002 % Zn.
dNlineral-Vitamin-premix contained 8.0% Mg (as oxide), 0.70% Fe (as sulfate), 2.40% S (as sulfate), 1.9 %
Cu (as sulfate), 6.0 % Mn (as oxide), 0.47 % I (as iodate), 0.075 % Se (as sodium selenite), 4.5 % Zn (as
oxide and sulfate), 133,363.4 IU/kg Vitamin A supplement, 412,272.7 IU/kg Vitamin D3 supplement, 259.1
IU/kg Vitamin E supplement, rice mill byproduct, and stabilized fat as a vitamin carrier.
eDry matter basis.









Table 4-2. Effect of dietary Al concentration and source on bone density of feeder
lambs as determined by radiographya
Treatmentc
1 2 3 4 5 6 SE
mm,
Day
28 5.71 4.78 6.15 5.48 6.07 4.90 0.55
69 5.76 6.14 6.60 6.91 6.49 6.22 0.44
109 4.96 5.03 5.01 5.48 6.44 6.08 0.67
aData represent least squares means, and pooled SE; across treatments; n = 7 per treatment
bDietary treatments were created by additions to a corn-SBM basal diet as follows: 1)
(Control) 10% sand; 2) 9.3% sand + 0.7% AlC13; 3) 7.5% sand, + 2.5% WTR; 4) 5% sand
+ 5%WTR; 5) 10% WTR; 6) 10% WTR + two times the added amount of mineral-vitamin
premix + 1.29% dicalcium phosphate; Treatments 2 and 3 were formulated to contain 2,000
ppm Al, treatment 4 was formulated to contain 4,000 ppm Al, and treatments 5 and 6 were
formulated to contain 8,000 ppm Al.
CMeans within rows did not differ (P < 0.05).




Table 4-3. Effect of dietary Al on dry fat-free bone mineral concentrations of Ca, P and
Mg for experimental diets
Treatment
1 2 3 4 5 6 SE
g/cm3C
Bone Density 1.89 1.91 1.94 1.89 1.93 1.93 0.003
mg /cm3-
P 74.5 76.1 75.2 81.1 81.1 78.3 5.9
Ca 209 209 197 211 211 191 1.3
Mg 4.13 3.72 4.08 4.24 3.78 3.89 0.07
%.d
Ash 69.9 68.8 69.1 68.9 68.9 68.9 1.56
P 14.2 14.4 14.6 15.4 14.1 15.1 0.63
Ca 39.9 39.4 38.1 39.5 37.0 37.7 18.3
Mg 0.79e 0.70ef 0.79e 0.79e 0.73ef 0.61f 0.04
aData represent least squares means and pooled SE; n = 7 per treatment
bDietary treatments were created by additions to a corn-SBM basal diet as follows: 1)
(Control) 10% sand; 2) 9.3% sand + 0.7% AlC13; 3) 7.5% sand, + 2.5% WTR; 4) 5% sand
+ 5%WTR; 5) 10% WTR; 6) 10% WTR + two times the added amount of mineral-vitamin
premix + 1.29% dicalcium phosphate; Treatments 2 and 3 were formulated to contain 2,000
ppm Al, treatment 4 was formulated to contain 4,000 ppm Al, and treatments 5 and 6 were
formulated to contain 8,000 ppm Al.
CCalculated using fresh weights
dCalculated using Ash weights
efMeans within rows lacking a common superscript differ (P < 0.05).














CHAPTER 5
SUMMARY AND CONCLUSIONS

In many developed nations, concerns about repeated application of manure to land

'has led to strict laws and regulations because of increased P levels in nearby water

bodies. Contamination with P can occur in sandy soils because of P leaching into the

ground water, and in slit or clay soils because of runoff of soluble P or erosion of P from

soil manure into local bodies of water. When P enters the water ways causes algae

growth is stimulated. When the algae die oxygen content of the water is decreased and

leads to aquatic plant and animal death.

A chemical reaction between Al and P binds soluble P making it unavailable as a

pollutant. During the water treatment process, Al is added to bind small particles, aiding

in the sedimentation processes, and resulting in the formation of water treatment residuals

(WTR). Water treatment residuals contain a nonavailable form of Al known to reduce P

runoff and leaching. Aluminum, when consumed in a bioavailable form, decreases

growth, intake, and body weight, and depresses bone deposition in several livestock

species. A major concern is that the Al in WTR, thought to be non-available, may

negatively impact an animal when ingested.

At the University of Florida, data were gathered to determine 1) if WTR are

harmful if ingested in amounts between 2,000 to 8,000 ppm Al and 2) determine the

availability of Al in WTR when compared to a control and a diet containing a

bioavailable form of Al (AlC13). A 111-d study was conducted to determine if declines in

intake, BW, ADG, bone mineral content (BMC), bone density, plasma P, tissue P, and









apparent P absorption were produced by the dietary administration of WTR to growing

lambs. Lambs were stratified by sex and randomly assigned to six dietary treatments; 1)

control (10% sand), 2) (9.7% sand and 0.3% A1C13), 3) (2.5% WTR and 7.5% sand), 4)

(5% WTR and 5% sand), 5) (10% WTR and 0% sand), and 6) (10% WTR, 0% sand, plus

double the quantities of the mineral-vitamin premix, and 1.29% dicalcium phosphate).

The WTR was analyzed to contain 7.8% Al on a DM basis. Ten percent of each diet was

either sand, WTR or the combination of the two. The diet concentrations of Al were 0,

2000, 4000, and 8000 ppm, respectively, for the six diets. Body weight, intake, and ADG

data were compared among three inclusion levels of Al as WTR, and one level of Al

from AlC13. Plasma samples and lamb weights were collected every 14 d. Fecal

collection (to determine apparent P absorption) occurred between d 91 and d 105, and

individual feeding occurred between d 91 and d 111. Samples of blood, brain, liver,

kidney, heart, and bone were collected upon experimental termination.

Lamb ADG, BW, and intakes were unaffected by dietary levels of WTR when

compared to the control. However, lambs fed 2,000 ppm Al from AlC13 had reduced

growth and lower ADG (P < 0.05) than other treatments.

Plasma P concentrations were unaffected by treatments at wk 0 or wk 2. The

control consistently had higher P concentrations than most other treatments, and the

WTR treatments generally had higher P concentrations than lambs given AlC13. Between

wk 6 and wk 14, plasma P concentrations began to increase after a decline at wk 6.

Currently, there is no evidence to explain this finding but this could be attributed to bone

mineral resorptions could have caused plasma P levels to increase and then stabilize.









Tissues were analyzed for concentrations of Ca, P, Mg, Cu, Fe, Mn, Se, and Zn.

Phosphorus concentrations were unchanged across treatments for all tissues and bone,

except kidney, where the control had a higher concentration of P than the lambs given

8,000 ppm Al form WTR (P < 0.05). Bone deposits of Mg were lowest for lambs fed

8,000 ppm Al from WTR with double the added mineral-vitamin premix, and 1.29%

dicalcium phosphate. All other bone mineral concentrations were unaffected by dietary

treatment. Iron concentrations were highest in the liver of lambs feed 8,000 ppm Al from

WTR and lowest in the controls (P < 0.05). Aluminum varied in most tissues, but brain

is the primary repository for Al and is the focus of much research. Concentrations of Al

in the brain were highest for animals receiving 2,000 ppm Al from AlC13 and lowest

lambs given 2,000 ppm Al from WTR (P < 0.05). Concentration, of Al increased when

Al from WTR was given in amounts above 2,000 ppm. The accumulation of Al in the

brain has not been shown to be a threat to the animal and cannot be critiqued without

further research.

On d 91, lambs were placed in metabolism crates; feed and feces were collected for

the determination of apparent P absorption. No differences in apparent P absorption were

observed among WTR treatments, however the lambs administered 2,000 Al via AlC13

had reduced apparent absorption of P. We conclude that Al in WTR does not interfere

with the apparent absorption of dietary P, but AlC13 causes apparent fecal P absorption to

decline.

There were no BMC and bone density differences in long bones collected from

lambs in any treatment. This lack of differences among treatments perhaps may be









attributed to the short duration of the study and because that bone mineral resorption had

not yet occurred in the long bones.

Based on the data collected during this trial and from previous studies, it can be

determined that 2,000 ppm Al via AlC13 results in poor animal performance and P tissue

and plasma P declines. However, intakes, BW and ADG of lambs receiving WTR in

amounts from 2,000 ppm to 8,000 ppm Al did not differ from the control. Thus, WTR

does not appear to negatively affect performance of growing sheep. The apparent P

absorption data strengthens the idea that Al in WTR is less available to the animal then

the Al in AlC13. Apparent P absorption was not altered in lambs fed WTR, but animals

fed 2,000 ppm AlC13 were negatively impacted. Additionally, plasma P and tissue

mineral levels, with the exception of brain Al, were not altered with the administration of

Al from WTR. Under these experimental conditions, dietary administration of Al from

WTR did not cause physiological tissue damages. Overall, it has been demonstrated that

Al from WTR does not negatively impact a growing lamb's health or performance and

could be administered at levels as high as 8,000 ppm Al without causing detrimental

effects. Additional research in other ruminant species should be conducted before data

can be proper applied to species other than the ovine.















APPENDIX
TABLE DATA

Table A-1. Effect of dietary Al concentration and source on ADG of feeder lambsa
Treatment

1 2 3 4 5 6 SE
ADG, g
Wk
2 125cd 9.3c 107cd 235cd 344d 134cd 109
4 116d 4.6c 227d 97.3cd 272d 116cd 68.7
6 274c 50.6df 213cd 250ce -6.4ef -32.4f 64.9
11 257de 287d 570c 593c 480cd 463ce 91.5
14 366 361 244 208 207 269 69.7
aData represent least squares means; n = 7 per treatment
bDietary treatments were created by additions to a corn-SBM basal diet as follows: 1)
(Control) 10% sand; 2) 9.3% sand + 0.7% AIC13; 3) 7.5% sand, + 2.5% WTR; 4) 5% sand
+ 5%WTR; 5) 10% WTR; 6) 10% WTR + two times the added amount of mineral-vitamin
premix + 1.29% dicalcium phosphate; Treatments 2 and 3 were formulated to contain 2,000
ppm Al, treatment 4 was formulated to contain 4,000 ppm Al, and treatments 5 and 6 were
formulated to contain 8,000 ppm Al.
cdefMeans within rows lacking a common superscript differ (P < 0.05).
















LITERATURE CITED


Ammerman, C.B., R. Valdivia, I.V. Rosa, P.R. Henry, J.P. Feaster, and W.G. Blue. 1984.
Effect of sand or soil as a dietary component on phosphorus utilization by sheep. J.
Anim. Sci. 58:1093-1099.

Basta, N.T., R.J. Zupancic, and E.A. Dayton. 2000. Evaluating soil tests to predict
bermuda grass growth in drinking water treatment residuals with phosphorus
fertilizer. J. Environ. Qual. 29:2007-2012.

Brady, N.C., and Weil, R.R. 2002. The Nature and Properties of Soils. Pearson
Education, Inc.

Capdevielle, M.C., L.E. Hart, J. Goof, and C.G. Scanes.1998. Aluminum and acid effects
on calcium and phosphorus metabolism in young growing chickens (Gallus gallus
domesticus) and mallard ducks (Anasplatyrhynchos). Arch. Environ. Contam.
Toxicol. 35:82-88.

Chappard, D., P. Insalaco, and M. Audran. 2003. Aluminum osteodystrophy and celiac
disease. Calcif. Tissue. Int. 10:223-26.

Cox, K.A. and M.A. Dunn. 2001. Aluminum toxicity alters the regulation of calbindin-
D28k protein and mRNA expression in chick intestine. J. of Nutr. 131:2007-2013.

Dayton, E.A., and N.T. Basta. 2001 Characterization of drinking water treatment residual
for use as a soil constituent. Water Environ. Res. 73:52-57.

Dayton, E.A., N.T. Basta, C.A. Jakober, and J.A. Hattey. 2003. Using treatment residuals
to reduce phosphorus in agriculture runoff. Am. Water Works Assoc. J.95:151-
159.

Elliott, H.A., G.A. O'Connor, P. Lu, and S. Brinton. 2002. Influence of water treatment
residuals on P solubility and leaching. J. Environ. Qual. 31:1362-1369.

Farm Press. 2004. Right phosphorus management can cut vegetable costs, runoff.
PRIMEDIA Business Magazine & Media Inc. Tampa, FL Feb. 7. p4.









Federal Register. 2004. Agency information collection activities; submission to OMB for
review and approval; comment request; national pollutant discharge elimination
system (NPDES) compliance assessment/certification information (renewal), EPA
ICR Number 1427.07, OMB Control Number 2040-0110. May 24. Vol. 69, no.
100.

Fethiere, R., R.D. Miles, and R.H. Harms. 1990. Influence of synthetic sodium
aluminosilicate on laying hens fed different phosphorus levels. Poult. Sci.
69:2195-2208.

Field, A.C. and D. Purves. 1964. The intake of soil by grazing sheep. Proc. Nutr. Soc.
23:24-25.

Gomez- Alonso,C. P. Menendez-Rodriguez, M.J. Virgos-Soriano, J.L. Fernandez-Martin,
M.T. Ferandez-Coto, and J.B. Cannata-Andia. 1996. Aluminum-induced
osteogensis in osteopenic rats with normal renal functions. Calcif. Tissue Int.
64:534-541.

Harris, W.D. and P. Popat. 1954. Determination of phosphorus content of lipids. Amer.
Oil Chem. Soc. J 31:124-126.

Haustein, G.K., T.C. Daniel, D.M. Miller, P.A. Moore, Jr., and R.W. McNew. 2000.
Aluminum-containing residuals influence high-phosphorus soils and runoff water
quality. J. Environ. Qual. 29:1954-1959.

Healy W.B. 1967. Ingestion of soil by sheep. Proc. New Zealand Soc. Anim. Prod.
27:109-115.

Healy W.B. 1968. Ingestion of soil by dairy cows. New Zealand J. Agr. Res. 11:487-
490.

Huff, W.E., P.A. Moore Jr., J.M. Balog, G.R. Bayyari, and N.C. Rath. 1996. Evaluation
of toxicity of aluminum in younger broiler chickens. Poult. Sci. 75:1359-1365.

Ippolito, J.A., K.A. Barbarick, D.M. Heil, J.P. Chandler and E.F. Redente. 2003.
Phosphorus retention mechanism of water treatment residuals. J. Environ. Qual.
32:1857-1864.

Kleinman, P.J.A., A.N. Sharpley, B.G. Moyer, and G.F. Elwinger. 2002. Effect of
mineral and manure phosphorus sources on runoff phosphorus. J Environ. Qual.
31:2026-2033.

Lanyon, L.S. 1994. Dairy manure and plant nutrient management issues affecting water
quality and the dairy industry. J. Dairy Sci. 77:1999-2007.

Lorentzen, A. 2004. Environmental group says Iowa not enforcing laws. The Associated
Press State and Local Wire, May 20.









Mann, R.A., and B.S. Roberts. 2000. Smiths and Roberts Business Law. 11th Ed. West
Legal Studies in Business, Thomson Learning, Cincinnati, OH. pp. 997-1002.

Martin, L.C., A. J. Clifford, and A.D. Tillman. 1969. Studies on sodium bentonite in
ruminant diets containing urea. J. Anim. Sci. 29:777-778.

McDowell, L.R. 2003. Minerals in Animal and Human Nutrition. 2nd Ed. Elsevier Sci.,
Amsterdam.

McDowell, L.R. 1997. Minerals for Grazing Ruminants in Tropical Regions. 3rd Ed. Bull.
animal science department University of Florida, Gainesville.

Meakim, D.W., E.A. Ott, R.L. Asquith and J.P. Feaster. 1981. Estimation of mineral
content of the equine third metacarpal by radiographic photometry. J. Anim. Sci.
53:1019-1026.

Meyer, D. 2000. Dairying and the environment. J. Dairy Sci. 83:1419-1427.

Miles, P.H., N.S. Wilkinson, and L.R. McDowell. 2001. Analysis of mineral for animal
nutrition research 3rd ed. University of Florida, Gainesville, FL.

Miller W.J. 1983. Phosphorus-ruminant-nutritional requirements, biochemistry and
metabolism. National Feed Ingredient Association's Mineral Ingredient Handbook.
NFIA, West Des Moines, Iowa. pp.1-14.

NRC (National Research Council)S. 1985. Nutrient Requirements of Domestic Animals.
Nutrient Requirements of Sheep, 5th Ed. Natl. Acad. Sci. Washington DC.

Novak, J.M. and D.W. Watts. 2004. Increasing the phosphorus sorption capacity of
southeastern coastal plain soils using water treatment residuals. Soil Sci. 169:206-
214.

National Research Council. 2000. Watershed Management for Potable Water Supply.
Natl. Acad. Press. Washington, DC.

O'Connor, G.A., H.A. Elliott, and P. Lu. 2002. Characterizing water treatment residuals
for P retention. Soil Crop Sci. Soc. Florida Proc. p67-73.

Ott, E.A., L.A. Lawrence, and C. Ice. 1987. Use of the image analyzer for radiographic
photometric estimation of bone mineral content. Proc. 10th Equine Nutr. Physiol.
Sym. Colorado State University. June 11-13.

Penn, C.J. and J.T. Sims. 2002. Phosphorus forms in biosolids amended soils, and losses
in runoff; effects of water treatment processes. J. Environ. Qual. 31:1349-1361.

Powers, W.J. 2003. Keeping science in environmental regulations: role of the animal
scientist. J. Dairy Sci. 86:1045-1051.









Rengel, Z. 2004. Aluminum cycling in the soil-plant-animal-human continuum.
BioMetals 17:669-689.

Rosa, V., P.R. Henry, and C.B. Ammerman. 1982. Interrelationship of dietary
phosphorus, aluminum and iron on performance and tissue mineral composition in
lambs. J. Anim. Sci. 55:1231-1240.

Rotz, C.A., A.N Sharpley, L.D. Satter, W.J Gburek, and M.A. Sanderson. 2002.
Production and feeding strategies for phosphorus management on dairy farms. J.
Dairy Sci. 85:3142- 3153.

Smith, D.R., P.A. Moore Jr, C.V. Maxwell, B.E. Haggar, and T.C. Daniel. 2004.
Reducing phosphorus runoff from swine manure with dietary phytase and
aluminum chloride. J Environ. Qual. 33:1048-1054.

Soon, Y.K. and T.E. Bates. 1982. Extractability and solubility of phosphate in soils
amended with chemically treated sewage sludges. Soil Sci. 134:89-96.

Tomas, F.M., and M. Somers. 1974. Phosphorus homeostasis in sheep. I. Effect of
ligation of parotid salivary ducts. Aust. J. Agric. Res. 25:475-483.

Underwood, E.J. and N.F. Suttle. 1999. The Mineral Nutrition of Livestock. 3rd Ed.
Midlothian, Wallingford, UK.

US Environmental Protection Agency (USEPA). 1997. Animal Waste Disposal Issues:
Office of Inspections, Washington. DC.

US Environmental Protection Agency (USEPA). 2003. Ecological soil screening level for
an interim final report. OERR, Washington DC.

Valdivia, R. 1977. Effect of dietary aluminum on phosphorus utilization by ruminants.
Ph.D. Dissertation, University of Florida, Gainesville.

Valdivia, R., C.B. Ammerman, P.R. Henry, J.P. Feaster, and C.J. Wilcox. 1982. Effect of
dietary aluminum and phosphorus on performance, phosphorus utilization and
tissue mineral composition in sheep. J. Anim. Sci. 55:402- 410.

Valdivia, R., C.B. Ammerman, C.J. Wilcox, and P.R. Henry. 1978. Effects of dietary
aluminum on animal performance and tissue mineral levels in growing steers. J.
Anim. Sci.47:1351-1360.

Water Resources. 2005. The official government website of Greensboro NC; water
treatment process. Greensboro, NC, http://www.greensboro-nc.gov/water/
supply/treatment.htm. Accessed: May 17, 2005.

Williams, S.N., L.A. Lawrence, L.R. McDowell, and N.S. Wilkinson. 1991a. Criteria of
evaluate bone mineral in cattle: II. Noninvasive techniques. J. Anim. Sci. 69:
1243-1254.









Williams, S.N., L.R. McDowell, A.C. Wamick, L.A. Lawrence, and N.S. Wilkinson.
1992. Influence of dietary phosphorus level on growth and reproduction of growing
beef heifers. Int. J. Anim.. Sci. 7:137-142.

Williams, S.N., L.R. McDowell, A.C. Wamick, L.A. Lawrence, and N.S. Wilkinson.
1990. Dietary phosphorus concentrations related to breaking load and chemical
bone properties in heifers. J. Dairy Sci. 73:1100-1106.

Williams, S.N., L.R. McDowell, A.C. Wamick, N.S. Wilkinson, and L.A. Lawrence.
1991b. Phosphorus concentrations in blood, milk, feces, bone and select fluids and
tissues of growing heifers as affected by dietary phosphorus. Liv. Res. for Rural
Dev. 3:67-79.

Williams, S.N., L.R. McDowell, A.C. Wamick, N.S. Wilkinson, and L.A. Lawrence.
1991c. Criteria of evaluate bone mineral in cattle: I. Effect of dietary phosphorus
on chemical, physical, and mechanical properties. J. Anim. Sci. 69:1232-1242.

Whetter, P.A., and D.E. Ullrey. 1978. Improved fluorometric method for determination
of selenium. J Assoc. Off. Anal. Chem. 4:927-930.

Zafar, T.A., D. Teegarden, C. Ashendel, M.A. Dunn, and C.M. Weaver. 2004.
Aluminum negatively impacts calcium utilization and bone calcium-deficient rats.
Nutr. Res. 24:243-259.














BIOGRAPHICAL SKETCH

Rachel Van Alstyne was born in Rochester, NY, on March 4, 1979, to Fred and

Andrea Van Alstyne. She was raised in the city of Rochester until the birth of her

brother, Timothy. At age seven, Rachel and her family moved to the suburbs of

Rochester, to the town ofFairport, NY.

From the age of 16, she maintained several jobs providing enough monetary

solidity to attain the education she desired after high school graduation. Rachel has

always had an incorrigible desire to be near animals. With the exception of her

employment at Brugger's Bagels as a shift manager, in all of her jobs she was able to

surround herself with animals and/or wildlife. Rachel's employment pursuits led her

from veterinary hospitals to the Seneca Park Zoo in Rochester, where she worked as an

animal care attendant better known as a "zoo keeper."

Rachel found it to be difficult during her first year as an undergraduate, since she

did not have the agricultural background many of her classmates did, but she persevered,

and maintained a near perfect GPA, proving that she was "cut out" for this lifestyle. She

desired enhancement in the field of animal care, and she began her pursuit to be a

veterinarian. During her junior year, Rachel decided that veterinary college was not on

the path for which she was best suited. She graduated from Cornell University with her

bachelor's degree in animal science in 2002. Upon graduation, she was undecided as to

which field she desired to pursue in graduate school, and began work. In 2003 Rachel

applied and was accepted to a master's program at the University of Florida with Dr. Lee






64


McDowell. After diligent days and evenings working in both the lab and barn, through

hurricanes, lost power, sick lambs, and inadequate staffing she was able to receive her

degree in 2005. While at the University of Florida she obtained a second master's degree,

concurrently, in management, from the Warrington College of Business. Unlike many

students, Rachel was unable to take even one semester to herself without enrollment in

classes. Her second degree in management from the college was both simulating and

cumbersome, but she was able to finish both degrees within a period of two years.