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Effect of protein supplementation on expression and distribution of urea transporter-B in lambs fed low-quality forage

P. A. Ludden^*, R. M. Stohrer^*, K. J. Austin^*, R. L. Atkinson^*, E. L. Belden and H. J. Harlow

^* Department of Animal Science, University of Wyoming, Laramie 82071 , Department of Veterinary Science, University of Wyoming, Laramie 82071 Department of Zoology and Physiology, University of Wyoming, Laramie 82071

ludden{at}uwyo.edu

Abstract

Two experiments were conducted to determine the effects of ruminal protein degradability, supplementation frequency, and increasing dietary protein on the expression and distribution of urea transporter-B (UT-B) in lambs fed low-quality forage (mature crested wheatgrass hay; 4.2 to 4.7% CP). In Experiment 1, 15 Dorset wether lambs (initial BW = 45.8 ± 1.3 kg) were blocked by initial BW and assigned to 1 of 3 treatments within a randomized complete block design for 28 d, with supplements fed to achieve 7, 10, or 13% total dietary CP. In Experiment 2, 13 Dorset wether lambs (initial BW = 34 ± 4 kg) were used in a completely randomized designed experiment and given 1 of 4 isonitrogenous supplements: 1) ruminally degradable protein (RDP) fed daily (n = 3), 2) RDP fed on alternate days (n = 3), 3) ruminally undegradable protein (RUP) fed on alternate days (n = 3), or 4) a 50:50 mixture of RDP and RUP fed on alternate days (n = 4) for 18 d. Alternate-day treatments were fed at twice that of daily supplementation. On the last day of both experiments, lambs were euthanized and samples taken for Western blot analyses for UT-B. Immunoblotting using a rabbit polyclonal antibody to UT-B confirmed the presence of distinct 32 kDa (consistent with a non-glycosylated UT-B protein) and 47 kDa (probable N-glycosylated form of UT-B) protein bands in all 9 tissues analyzed. In both experiments, the liver, dorsal rumen, reticulum, and ventral rumen displayed strong bands at 32 kDa and lighter bands at 47 kDa, whereas the cecum, large colon, spiral colon, and parotid salivary gland displayed slight 32 kDa bands and stronger, more visible bands at 47 kDa. Both protein bands were apparent in the kidney at similar visual intensities in Experiment 1, whereas the relative intensities of the 2 UT-B bands in the kidney were variable, and appeared somewhat reciprocal among animals. Although the abundance of the 47 kDa UT-B band in the ventral rumen was greater (P = 0.03) in lambs fed RDP daily in Experiment 2, no other treatment differences (P 0.15 to 0.99) in the abundance of the 32 or 47 kDa UT-B proteins within tissues were observed in either experiment. Although protein supplementation strategy had little effect on UT-B expression in tissues other than the ventral rumen, differences in the degree of glycosylation of UT-B across tissues may provide insight into its regulation.

Key Words: glycosylation • protein supplementation • rumen • salivary glands • sheep • urea transporter

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				P. Tickle, A. Thistlethwaite, C. P. Smith, and G. S. Stewart Novel bUT-B2 urea transporter isoform is constitutively activated Am J Physiol Regulatory Integrative Comp Physiol, August 1, 2009; 297(2): R323 - R329. [Abstract] [Full Text] [PDF]