Brief Summary of Minutes of Annual Meeting

Steven R. Alm (stevealm@uri.edu) - University of Rhode Island;
Paul Backman (pbackman@psu.edu) - Pennsylvania Agricultural Experiment Station;
Mark J. Carroll (mc92@umail.umd.edu) - University of Maryland;
J. Marshall Clark (jclark@ent.umass.edu) - University of Massachusetts;
Bruce B. Clarke (clarke@aesop.rutgers.edu) - Rutgers University;
Richard S. Cowles (rcowles@caes.state.ct.us) - Connecticut Agricultural Experiment Station;
J. Scott Ebdon (sebdon@pssci.umass.edu) - University of Massachusetts;
Steven Fales;
Karl Guillard (karl.guillard@uconn.edu) - University of Connecticut;
Richard J. Hull (rhu6441@postoffice.uri.edu) - University of Rhode Island;
Noel Jackson;
Peter J. Landschoot (pj11@psu.edu) - The Pennsylvania State University;
Pim Larsson-Kovach (il11@cornell.edu) - Cornell University;
James Lin - USEPA;
Bill Meyer (wmeyer@aesop.rutgers.edu) - Rutgers University;
Kevin Morris - NTEP;
Bridget Ruemmele (bridgetr@uri.edu) - University of Rhode Island;
Mike Sullivan (senmike@uriacc.uri.edu) - University of Rhode Island;
Patricia Vittum (pvittum@ent.umass.edu) - University of Massachusetts

Objective 1: To assess the autocrine, paracrine and endocrine mediation of humoral agents affecting proliferation, differentiation and metabolism in critical cells and tissues.

The Minnesota (MN) Station (ST) maintains with two genetically divergent populations of Holstein cows. One population (control line; CL) represents US breed average Holsteins in 1964 and a select line (SL) that represents contemporary US Holsteins. Using this model, the MN-ST has investigated hepatic gene expression of GH-R (GH-R1A) and IGF-I. Although expression of beta-actin was not altered by day postpartum (PP), expression of GAPDH and cyclophilin were reduced at 20 d PP and returned to prepartum levels at least by d 68 PP. A similar PP decrease and subsequent increase in expression of IGF-I and GH-R1A also occurred. Because beta-actin expression was not altered by day PP, parity or line, it was a more appropriate internal control than either GAPDH or cyclophilin for standardizing hepatic expression of GHR-1A and IGF. Selection for milk yield has reduced expression of GHR-1A in multiparous cows, has had no affect on hepatic IGF-BP3 or IGF-BP5 mRNA, and has tended to increase hepatic IGF-I mRNA during the periparturient period. In contrast, selection has decreased serum IGF-I. Collaborative efforts between the MN and Vermont (VT) ST have demonstrated greater expression of hepatic insulin receptor at -20 1 1 d PP in SL than CL cows. Expression of the insulin receptor did not differ between lines during early lactation.

The Maryland (MD) ST, with the Delaware (DL) ST, utilized hypothalami and pituitaries of broiler chickens from embryonic d 12 through post-hatch d 35. RNA was used for production and normalization of a cDNA library. The two most predominate cDNAs found were for proopiomelanocortin and GH. The unique cDNA inserts will be printed in DNA microarrays for analysis of mRNA levels in lines of chickens divergently selected for growth rate and body composition. The goal is to identify genes expressed in the hypothalamus or anterior pituitary that could account for differences in growth rate and body fat in chickens.

The New Jersey (NJ) ST, in collaboration with the New York (NY) ST, continues to investigate mechanisms by which the IGF system regulates growth of bovine mammary epithelial (BME) cells. MAC-T cells that constitutively express IGF binding protein-3 (IGFBP-3) exhibit enhanced DNA synthesis in response to IGF-I relative to mock-transfected control cells. This response is independent of a physical association between IGF-I and IGFBP-3, and appears to be related to activation of the IGF receptor signaling cascade. To determine the mechanism by which IGFBP-3 enhances the activity of IGF-I, the signal transduction pathways activated by IGF-I were determined in wild-type MAC-T cells. Following treatment with IGF-I, insulin-receptor substrate-1 was phosphorylated, leading to association with the p85 regulatory subunit of phosphatidylinositol 3-kinase (PI3K). IGF-I stimulated a time-dependent activation of Akt/PKB, a downstream effector of PI3K. IGF-I did not activate extracellular regulated-kinase (ERK) 1/2, suggesting that IGF-I does not stimulate DNA synthesis via this kinase pathway. In MAC-T cells that express IGFBP-3, phosphorylation of Akt following stimulation with IGF-I was enhanced relative to mock-transfected cells. Enhancement was detectable within 1 min of IGF-I treatment and persisted for up to 10 h. These findings suggest that IGFBP-3 may potentiate IGF action via enhanced activation of intracellular signaling molecules. They are also investigating mechanisms responsible for IGFBP-3 synthesis in BME cells. IGF-I and cAMP stimulate IGFBP-3 synthesis in these cells. Both enhance IGFBP-3 mRNA stability in MAC-T cells. A combination of IGF-I and cAMP increased IGFBP-3 promoter activity. Deletion analysis of the promoter indicated that a 200 bp region immediately upstream of the transcription start site may contain consensus binding elements that are responsible for this effect. Several potential binding sites for AP2 and Sp1 transcription factors have been proposed as potential mediators of the responsiveness to IGF-I and cAMP.

The Connecticut (CT) ST, with the MN ST, has quantified GH releasing factor (GRF)-induced GH secretion in cattle. They have attempted to quantify SRIF (somatostatin) inhibition of baseline GH and GRF-induced GH with little success. In these past experiments they have utilized the 14 amino acid form of SRIF. The experiments were repeated using SRIF 28 and demonstrated in heifers that SRIF 28 inhibits GRF-induced GH secretion and tends to reduce baseline GH concentrations. The MN ST is testing if GH response to GRF would be more repeatable after a preliminary GRF challenge.
Objective 2. To characterize the coordination of physiological actions of humoral factors that accommodate adaptation and influence the efficiency of nutrient utilization in food-producing animals.

The DL ST is establishing methods to quantify individual milk proteins in lactating dairy cows. They are using phase-gel electrophoresis to quantify the rate of production of several caseins, alpha-lactalbumin, and beta-lactoglobulin over the entire lactation. They are also using Capillary Zone Electrophoresis methods, that are more rapid and accurate, to analyze these proteins. This methodology is amenable to subsequent analysis of individual protein fractions by mass spectrometry if stable isotope infusion methods have been used. This will allow quantification of direct arterial contributions of amino acids to mammary protein synthesis and secretion, and facilitate estimation of rates of intra-mammary oxidation and transamination of amino acids.

The MD ST continues to study the regulation of somatotroph differentiation during chicken embryonic development. GH cell differentiation occurs by embryonic day (e) 16 in the chicken. They have reported that injection of corticosterone into chicken eggs on e11 induces premature GH cell differentiation on e13 or e14. ACTH was injected into fertile eggs on e9, e10, and e11. GH cell abundance was assessed on e14 using immunocytochemistry. ACTH treatment on e9, e10, and e11 was able to increase the number of GH cells, relative to water-injected and non-treated controls. Injection on e10 or e11 with 6x10-5 M, but not 6x10-6 M ACTH, increased GH cells on e14, indicating that 100 5l of 6x10-5 M ACTH was the minimum effective dose. GRF, alpha melanocyte-stimulating hormone (MSH), and ACTH were injected on e11, and GH cells were counted on e14. Although ACTH increased GH cells, relative to water-injected and non-treated controls, GRF and MSH failed to increase GH cells. They concluded that ACTH can prematurely increase the population of GH cells in the anterior pituitary gland. These findings suggest that the embryonic adrenal gland can produce sufficient corticosterone to play a role in regulating GH cell differentiation during chicken embryonic development.

The Maine (ME) ST is cloning the bPL (bovine placental lactogen) gene. They will use the bPL clone to produce bPL for physiological studies and autoimmunize heifers against bPL to understand the function of this compound. In collaboration with the MN ST, they have investigated at the relationship between bPL and IGF in the two lines of dairy cows at the MN ST. The ME and MN ST have examined the repeatability of concentrations of bPL in successive lactations. The ME ST, in collaboration with the California (CA) ST, is investigating the relationship between bPL and calf size in embryos from superovulation or in vitro fertilization procedures.

Collaborative studies between the MN ST and the Missouri ST continue. Cows from the genetic lines suggest reduced plasma progesterone and IGF-I concentrations may partially explain the reduced reproductive efficiency associated with high producing cows.

The CT ST, with the MN and Illinois ST, continue to investigate the ontogeny of the somatotropic axis in male (M) and female (F) beef cattle from birth to 1 yr of age. Serum concentrations of GH were greater in M than F. From birth to 1 yr, GH decreased in M and F. In M, IGF increased from birth to 186 d of age, but not in F. From birth to 165 d of age, BP-3 increased in M and F. BP-2 increased in F but not in M in the same time frame. The MN ST is using heifer calves to examine the role of hepatic GHR-1A and IGF-I during periods of normal (ADG = .8 kg/d), restricted (50% of maintenance requirement), and enhanced (compensatory) growth. Relationships among genetic merit, concentrations of IGF-I and GH, and hepatic expression of GH-R1A and IGF-I are being evaluated.

The CT ST continues to study the interaction of zinc (Zn), thyroid hormone (T3) and the somatotropic axis. Concentrations of IGF averaged 705, 1072 and 1445 ng/mL and 752, 989 and 1509 ng/mL in Zn deficient, pair fed and control rats and hypo, hyper and euthyroid animals, respectively. Zn status, but not T3 influenced IGFBP 3. IGFBP-3 in Zn-D and PF was less than C, but were not different from each other. There was no effect of treatments IGFBP-2 concentrations. Thus, Zn and T3 status altered T3 and IGF concentrations but only Zn status affected IGFBP-3 levels.

The MN ST demonstrated that plasma IGF-I concentrations in SL cows were less than in CL cows during wk 2 through 7 of lactation. Energy balance of the SL and CL cows was similar during this interval. This difference was related to milk yield as a similar though less significant relationship was observed when SL cows were separated into low, medium and high producers. One hypothesis for this difference is an increased clearance rate of IGF-I from the plasma of high producing cows.

The MN ST is testing the effect of season on success of induction of lactation was assessed with multiparous Holstein reproductive cull cows. Cows were induced in February or June with twice daily subcutaneous injections of estradiol and progesterone for 7 d, twice daily mammary massage for the next 6 d, and dexamethasone on d 13 of study. Cows received bST on d 0, 10, 20, and 30 of study and at 14 d intervals thereafter. Milking (3x/d) commenced on d 14 and continued for 122 d. Induction was successful in 85% of the cows and milk yield was not affected by season. Inclusion of bST in the treatment scheme may partially offset the negative effect of short day length on milk yield from induced cows.

Body composition and D2O dilution data from five studies (Washington, CA, CT and MN ST and laboratories in France) are being pooled in an attempt to develop more universally applicable prediction equations based on D2O dilution.

Objective 3. To develop integrative concepts that describe the regulation of nutrient utilization and result in enhanced biological efficiency in the production of high-quality products to meet changing consumer demands.

With the addition of individuals at the MD and DL ST, members of NE-148 can begin to supply them with empirical data to begin to develop mechanistic models of growing and lactating cattle. The DL ST is developing models of the lactating dairy cow, with emphasis on protein metabolism, synthesis and secretion by the mammary gland. They are refining previous models that describe amino acid transport across membranes within the gland, as well as individual milk protein transcription and translation. The goal is to produce a model that gives reasonable predictions of mammary substrate absorption from arterial blood and milk component output. Model development is done in a way as to produce a general model structure that is transferable to all tissues within the lactating dairy cow. A complete cow model can be produced from tissue models that are compatible. Each individual component model can thus be updated individually as new data and information become available.

Auchtung T.L., D.A. Buchanan, C.A. Lents, S.M. Barao, and G.E. Dahl. 2001. Growth hormone (GH) response to growth hormone-releasing hormone (GHRH) in beef cows divergently selected or milk production. J. Anim. Sci.79:1295-1300.