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Lysine supply is potentially limiting for milk production in dairy cows. The availability of Lys to the mammary gland and other tissues is a function of the quantity of metabolizable Lys supplied and Lys catabolism by the liver. Likewise, Lys catabolism may be influenced by Lys supply. This study evaluated the effect of increased postruminal Lys supply on the expression of aminoadipate semialdehyde synthase (AASS, a committing step in Lys catabolism in the liver) and ornithine transcarbamoylase and argininosuccinate synthase (key urea cycle enzymes that are responsive to protein supply). Eight multiparous peak Holstein cows were used in a replicated 4 × 4 Latin square. Cows were fed a Lys-limiting ration and infused postruminally with 0, 9, 27, or 63 g/d of Lys. The study consisted of 10 d of pretreatment followed by 10 d of Lys infusion. On the last day of each period, liver and milk samples were collected for mRNA analysis, and blood samples were collected for analysis of amino acids and Lys metabolites. Milk protein percent increased by 5.9%, plasma Lys increased by 74%, and α-aminoadipic acid increased by 51% with postruminal infusion of 63 g/d Lys compared with 0 g/d. Expression of AASS, ornithine transcarbamoylase, and argininosuccinate synthase mRNA in liver did not differ with postruminal infusion of Lys. Milk fat globule mRNA for major milk proteins and AASS were not affected by Lys infusion. Postruminal infusion of Lys resulted in an 86% greater increase in AASS mRNA in the liver compared with mammary mRNA. These changes suggest that hepatic Lys metabolism is not responsive to Lys supply at the transcription level, and that the availability of Lys to extrahepatic tissue may be determined by hepatic Lys metabolism.

Two independent studies were conducted to determine whether mechanical mixing of total mixed ration (TMR) or TMR dry matter alters Lys release from 6 rumen-protected Lys (RPL) products (A, B, C, D, E, and F). In the first study, routine mixing procedures were simulated to determine if inclusion of RPL products in TMR altered in situ release of Lys. Following mixing, Dacron bags containing RPL products were ruminally incubated for 0, 6, 12, or 24 h to determine Lys release. The second study occurred independently of the first, in which Lys release from RPL products was evaluated when incorporated into a TMR that differed in dry matter (DM) content. Bags containing TMR and RPL product mixture were stored at room temperature for 0, 6, 18, and 24 h to simulate RPL product exposure to TMR when mixed and delivered once per day. Concentration of free Lys in both studies was determined using ultra-performance liquid chromatography. Following mechanical mixing, ruminal Lys release was significantly greater for C and tended to increase for F. Mechanical mixing did not alter ruminal Lys release from other RPL products evaluated. Hours of ruminal incubation significantly altered Lys release for all products evaluated, and a significant interaction of mechanical mixing and hours of ruminal incubation was observed for A and C. Exposure to lower TMR DM (40.5 versus 51.8%) significantly increased Lys release from B but did not alter Lys release from the other RPL products evaluated. Moreover, time of exposure to TMR significantly increased Lys release from all RPL products evaluated, and a significant interaction of TMR DM and time of exposure to TMR was observed for B and E. These data suggest mechanical mixing and variation in TMR DM may compromise the rumen protection of RPL products; therefore, on-farm feeding practices may alter efficacy of RPL products in dairy rations.

The objective of this experiment was to measure ruminal and lactational responses of Holstein dairy cows fed diets containing 3 different starch levels: 17.7 (low; LS), 21.0 (medium; MS), or 24.6% (high; HS). Twelve multiparous cows (118 ± 5 d in milk) were assigned randomly to dietary treatment sequence in a replicated 3 × 3 Latin square design with 3-wk periods. All diets were fed as total mixed rations and contained approximately 30.2% corn silage, 18.5% grass silage, and 5.0% chopped alfalfa hay. Dietary starch content was manipulated by increasing dry ground corn inclusion (% of dry matter) from 3.4 (LS) to 10.1 (MS) and 16.9 (HS) and decreasing inclusion of beet pulp and wheat middlings from 6.7 and 13.4 (LS) to 3.4 and 10.1 (MS) or 0 and 6.8 (HS). In vitro 6-h starch digestibility of the diet increased as nonforage sources of fiber replaced corn grain (% of dry matter; 73.6, HS; 77.3, MS; 82.5, LS) resulting in rumen-fermentable starch content by 14.6, 16.2, and 18.1% for the LS, MS, and HS diets, respectively. Diets had similar neutral detergent fiber from forage and particle size distributions. Dry matter intake, solids-corrected milk yield, and efficiency of solids-corrected milk production were unaffected by diet, averaging 26.5 ± 0.8, 40.8 ± 1.6, and 1.54 ± 0.05 kg/d, respectively. Reducing dietary starch did not affect chewing time (815 ± 23 min/d), mean ruminal pH over 24h (6.06 ± 0.12), acetate-to-propionate ratio (2.4 ± 0.3), or microbial N synthesized in the rumen (585 ± 24 g/d). Total tract organic matter digestibility was higher for HS compared with MS and LS diets (69.2, 67.3, and 67.0%, respectively), but crude protein, neutral detergent fiber, and starch digestibilities were unaffected. As dietary starch content decreased, in vitro ruminal starch fermentability increased and, consequently, the range between HS and LS in rumen-fermentable starch (3.5 percentage units) was less than the range in starch content (6.9 percentage units). Under these conditions, dietary starch content had no measurable effect on ruminal fermentation or short-term lactational performance of high-producing Holstein dairy cows.

Milk urea nitrogen (MUN) is correlated with N balance, N intake, and dietary N content, and thus is a good indicator of proper feeding management with respect to protein. It is commonly used to monitor feeding programs to achieve environmental goals; however, genetic diversity also exists among cows. It was hypothesized that phenotypic diversity among cows could bias feed management decisions when monitoring tools do not consider genetic diversity associated with MUN. The objective of the work was to evaluate the effect of cow and herd variation on MUN. Data from 2 previously published research trials and a field trial were subjected to multivariate regression analyses using a mixed model. Analyses of the research trial data showed that MUN concentrations could be predicted equally well from diet composition, milk yield, and milk components regardless of whether dry matter intake was included in the regression model. This indicated that cow and herd variation could be accurately estimated from field trial data when feed intake was not known. Milk urea N was correlated with dietary protein and neutral detergent fiber content, milk yield, milk protein content, and days in milk for both data sets. Cow was a highly significant determinant of MUN regardless of the data set used, and herd trended to significance for the field trial data. When all other variables were held constant, a percentage unit change in dietary protein concentration resulted in a 1.1mg/dL change in MUN. Least squares means estimates of MUN concentrations across herds ranged from a low of 13.6 mg/dL to a high of 17.3 mg/dL. If the observed MUN for the high herd were caused solely by high crude protein feeding, then the herd would have to reduce dietary protein to a concentration of 12.8% of dry matter to achieve a MUN concentration of 12 mg/dL, likely resulting in lost milk production. If the observed phenotypic variation is due to genetic differences among cows, genetic choices could result in herds that exceed target values for MUN when adhering to best management practices, which is consistent with the trend for differences in MUN among herds.

Physiological effects of thyroid hormones are mediated primarily by binding of triiodothyronine to specific nuclear receptors. Organ-specific changes in production of triiodothyronine from its prohormone, thyroxine, have been hypothesized to target the action of thyroid hormones on the mammary gland and play a role in mediating or augmenting a galactopoietic response to bovine somatotropin (bST). Additionally, tissue responsiveness to thyroid hormones may be altered by changes in the number or affinity of nuclear receptors for thyroid hormones. In the present study, effects of bST and bovine growth hormone-releasing factor (bGRF) on thyroid hormone receptors in liver and mammary gland were studied. Lactating Holstein cows received continuous infusions of bST or bGRF for 63 d or served as uninfused controls. Nuclei were isolated from harvested mammary and liver tissues and incubated with [(125)I]-triiodothyronine. Treatments did not alter the capacity or affinity of specific binding sites for triiodothyronine in liver or mammary nuclei. Evaluation of transcript abundance for thyroid hormone receptors showed that isoforms of thyroid hormone receptor or retinoid receptor (which may influence thyroid receptor action) expressed in the mammary gland were not altered by bST or bGRF treatment. Data do not support the hypothesis that administration of bST or bGRF alters sensitivity of mammary tissue by changing expression of thyroid hormone receptors.

Our objective was to determine if prolonged exposure to various durations of light (i.e., photoperiod) would affect serum IGF-I concentrations in heifers. Thus, 16 Holstein heifers, 2 to 4 mo of age, were exposed during a 5-wk pretreatment period to 12 h of cool-white fluorescent light (L) at an intensity of 1200 lux and 12 h of dark (D). At 5 wk, photoperiods were adjusted to 24 L: 0 D (24 L), 20 L: 4 d (20 L), 16 L: 8 D (16 L) or 8 L: 16 D (8 L) per 24 h (n=4 heifers per photoperiod treatment). Blood was sampled at 5 wk and monthly for 4 mo. During each sampling period, blood was collected at 16-h intervals for 48 h and serum IGF-I was determined by RIA. Photoperiod treatment, month of experiment and their interactions affected serum IGF-I concentrations. Averaged over months, concentrations of serum IGF-I was greatest in heifers on 16 L; heifers on 20 L had IGF-I concentrations similar to 8 L, 16 L and 24 L and heifers on 24 L had concentrations similar to that of heifers on 8 L. Heifers in all treatment groups exhibited an increase in serum IGF-I concentration during the 4 mo of treatment. Heifers on 16 L and 20 L exhibited the greatest difference in serum IGF-I concentrations compared with 8L heifers after 3 mo of treatment. In conclusion, 16 L increases concentrations of serum IGF-I above that seen for heifers treated with 8 L or 24 L.

Serotonin stimulates secretion of growth hormone (GH) in cattle, but the mechanism is unknown. In rats, thyrotropin-releasing hormone (TRH) mediates serotonin-induced secretion of GH. We hypothesized that the same is true in cattle. Cattle were fed for 2h daily to synchronize secretion of GH, such that concentrations of GH were high before and low after feeding. Our first objective was to determine whether or not feeding suppresses serotonin receptor agonist (quipazine) induced secretion of GH. Holstein steers were injected with quipazine (0.2 mg/kg BW) either 1 h before or 1 h after feeding. Quipazine-induced secretion of GH which did not differ in magnitude before and after feeding. If TRH mediates serotonin-induced secretion of GH, then magnitude of TRH-induced secretion of GH should not be different before and after feeding (our second objective). Sixteen meal-fed Holstein steers were injected with 0.3 microg TRH/kg BW either 1 h before or 1 h after feeding. Indeed, magnitude of TRH-induced secretion of GH before and after feeding was not different. Our third objective was to inhibit endogenous TRH with 3,5,3'-triiodothyronine (T(3)) and examine basal, GH-releasing hormone (GHRH)-, TRH- and quipazine-induced secretion of GH. Sixteen Holstein steers were injected daily with either T(3) (3 or 6 microg/kg BW) or vehicle for 20 days and then challenged sequentially with vehicle or GHRH, TRH, or quipazine. T(3) did not affect basal, GHRH- or TRH-induced secretion of GH, but reduced basal secretion of thyroxine. T(3) reduced but did not completely block quipazine-induced secretion of GH. In conclusion, TRH mediates, in part, serotonin-induced secretion of GH in cattle.

Pituitary adenylate cyclase-activating polypeptide (PACAP) is a hypothalamic neuropeptide that stimulates release of growth hormone (GH) from cultured bovine anterior pituitary gland cells, but the role of PACAP on the regulation of in vivo secretion of GH in cattle is not known. To test the hypothesis that PACAP induces secretion of GH in cattle, meal-fed Holstein steers were injected with incremental doses of PACAP (0, 0.1, 0.3, 1, 3, and 10 microg/kg BW) before feeding and concentrations of GH in serum were quantified. Compared with saline, injection of 3 and 10 microg PACAP/kg BW increased peak concentrations of GH in serum from 11.2 ng/ml to 23.7 and 21.8 ng/ml, respectively (P0.01). Peak concentrations of GH in serum were similar in steers injected with 3 or 10 microg PACAP/kg BW. Meal-fed Holstein steers were then injected with 3 microg/PACAP/kg BW either 1 hr before feeding or 1 hr after feeding to determine if PACAP-induced secretion of GH was suppressed after feeding. Feeding suppressed basal concentrations of GH in serum. Injection of PACAP before feeding induced greater peak concentrations of GH in serum (19.2 +/- 2.6 vs. 11.7 +/- 2.6 ng/ml) and area under the response curve (391 +/- 47 vs. 255 +/- 52 ng. ml(-1) min) than injection of PACAP after feeding, suggesting somatotropes become refractory to PACAP after feeding similar to that observed by us and others with growth hormone-releasing hormone (GHRH). We concluded that PACAP induces secretion of GH and could play a role in regulating endogenous secretion of GH in cattle, perhaps in concert with GHRH.

Growth hormone (GH) is essential for postnatal somatic growth, maintenance of lean tissue at maturity in domestic animals and milk production in cows. This review focuses on neuroregulation of GH secretion in domestic animals. Two hormones principally regulate the secretion of GH: growth hormone-releasing hormone (GHRH) stimulates, while somatostatin (SS) inhibits the secretion of GH. A long-standing hypothesis proposes that alternate secretion of GHRH and SS regulate episodic secretion of GH. However, measurement of GHRH and SS in hypophysial-portal blood of unanesthetized sheep and swine shows that episodic secretion of GHRH and SS do not account for all episodes of GH secreted. Furthermore, the activity of GHRH and SS neurons decreases after steers have eaten a meal offered for a 2-h period each day (meal-feeding) and this corresponds with reduced secretion of GH. Together, these data suggest that other factors also regulate the secretion of GH. Several neurotransmitters have been implicated in this regard. Thyrotropin-releasing hormone, serotonin and gamma-aminobutyric acid stimulate the secretion of GH at somatotropes. Growth hormone releasing peptide-6 overcomes feeding-induced refractoriness of somatotropes to GHRH and stimulates the secretion of GHRH. Norepinephrine reduces the activity of SS neurons and stimulates the secretion of GHRH via alpha(2)-adrenergic receptors. N-methyl-D,L-aspartate and leptin stimulate the secretion of GHRH, while neuropeptide Y stimulates the secretion of GHRH and SS. Activation of muscarinic receptors decreases the secretion of SS. Dopamine stimulates the secretion of SS via D1 receptors and inhibits the secretion of GH from somatotropes via D2 receptors. Thus, many neuroendocrine factors regulate the secretion of GH in livestock via altering secretion of GHRH and/or SS, communicating between GHRH and SS neurons, or acting independently at somatotropes to coordinate the secretion of GH.

When I was a beginning graduate student 41 yr ago it had been established that estrogen caused mammary duct growth; a combination of estrogen and progesterone was required for lobule-alveolar development of the mammary glands; and prolactin and growth hormone were essential for mammary growth. In laboratory species exogenous prolactin, glucocorticoids, and estrogen would initiate secretion of milk provided the mammary glands had a well-developed lobule-alveolar system. It was not known with certainty that progesterone inhibited the process. For some species, prolactin and thyroxine had been shown to stimulate lactation, while glucocorticoids suppressed lactation. Definitive roles for growth hormone and insulin during lactation had not been established. Studies of hormonal control of mammary growth and function in cattle were few. In vitro methods to study hormonal regulation of the mammary glands were in their infancy. Quantitative measures of changes in mammary cell numbers and specific components of milk in response to hormones were rare. The concepts for quantification of hormone concentrations, hormone receptors, growth factors, and binding proteins in blood; hormonal regulation of nutrient partitioning; and hormonally induced mechanisms of action within mammary cells were waiting to be discovered. And eventually they were. However, lest we become too enamored with our current understanding of the hormones that control mammary growth and lactation, it remains a fact that the greatest physiological stimulus for milk yield is pregnancy, not some cocktail of exogenous hormones, growth factors, receptor agonists/ antagonists, or gene therapies. Viva la mom!

The objective of this study was to evaluate the effects of recombinant bovine growth hormone-releasing factor (rGRF) or recombinant bovine somatotropin (rbST) on growth and function of the first-wave dominant follicle and corpus luteum. Primiparous Holstein cows (117 d postpartum) were infused with 12 mg/d of rGRF (n = 10) or 29 mg/d of rbST (n = 10) for 63 d, and non-infused cows (n = 10) were controls. At slaughter on d 5 of an estrous cycle, blood and ovaries were collected and data from cows with a corpus luteum were analyzed (control, n = 8; rGRF, n = 5; rbST, n = 6). Treatment with rGRF or rbST increased somatotropin (ST) and IGF-I in serum similarly compared with controls. In contrast, rbST-treated cows had higher concentrations of ST in follicular fluid (FF) compared with rGRF-treated and control cows. In addition, rbST, but not rGRF, increased the number and decreased the size of estrogen-active follicles (EA; estradiol > progesterone concentrations in FF), increased the abundance of IGF binding proteins-2, -3, and -4 in FF from EA follicles, and increased the number but decreased the size of corpora lutea and decreased concentration of progesterone in serum compared with controls. Based on these results, we concluded that long-term infusion of rbST alters growth and function of the first-wave dominant follicle and the corpus luteum in cattle.

The selective dopamine D1 receptor agonist, SKF38393, stimulates release of somatostatin (SS) from perifused bovine hypothalamic slices. Therefore, we hypothesized that SKF38393 activates SS neurons, which, via release of SS, would suppress concentrations of growth hormone (GH) in serum in calves. Our objectives were to determine whether SKF38393: (1) increases the percent of immunoreactive c-Fos protein and Fos-related antigens (Fos/FRA) detected in somatostatin neurons in periventricular (PeVN) and arcuate (ARC) hypothalamic nuclei; (2) reduces concentrations of GH in serum; (3) suppresses growth hormone-releasing hormone (GHRH)-induced release of GH. Meal-fed steers were used to perform these objectives because a synchronous pulse of GH occurs 1-2 hr before feeding in steers allowed access to feed for 2 hr each day. In Experiment 1, two groups of four Holstein steers were injected s.c. with either vehicle (sterile water) or SKF38393 (5 mg/kg BW). Steers were injected i.v. with a lethal dose of sodium pentobarbital 100 min later and their brains were fixed with 4% paraformaldehyde. Dual-label immunohistochemistry was performed on 40 microns free-floating sections using antiserum to SS and to Fos/FRA on sections containing PeVN and ARC nuclei. More SS neurons were detected in the PeVN than in the ARC. The percent of SS neurons with immunoreactive Fos/FRA present was 2.9-fold higher in SKF38393-treated compared with vehicle-injected steers in the PeVN, but was unchanged in the ARC. In Experiment 2, eight Holstein steers were injected s.c. with either vehicle (sterile water) or SKF38393 (5 mg/kg BW) 140 min before meal-feeding. In contrast to controls, concentrations of GH in serum of SKF38393-treated steers did not increase during 140 min before meal-feeding. In Experiment 3, eight Holstein steers were injected s.c. with either vehicle (sterile water) or SKF38393 (5 mg/kg BW), then 100 min later, each steer was injected i.v. with [Leu27,Hse45] bGHRH1-45 lactone (0.2 micrograms/kg BW). Bovine GHRH stimulated release GH into serum in both groups, but concentrations of GH were lower in SKF38393-treated steers. These results show that stimulation of D1 receptors selectively increases activity of SS neurons in the PeVN, and this increased activity is associated with suppressed basal- and GHRH-induced release of GH in serum of meal-fed steers.

This experiment evaluated the effect of feeding a lower starch diet (21% of dry matter) with different amounts of forage (52, 47, 43, and 39% of dry matter) on lactational performance, chewing activity, ruminal fermentation and turnover, microbial N yield, and total-tract nutrient digestibility. Dietary forage consisted of a mixture of corn and haycrop silages, and as dietary forage content was reduced, chopped wheat straw (0–10% of dry matter) was added in an effort to maintain chewing activity. Dietary concentrate was adjusted (corn meal, nonforage fiber sources, and protein sources) to maintain similar amounts of starch and other carbohydrate and protein fractions among the diets. Sixteen lactating Holstein cows were used in replicated 4×4 Latin squares with 21-d periods. Dry matter intake increased while physically effective neutral detergent fiber (peNDF 1.18 ) intake was reduced as forage content decreased from 52 to 39%. However, reducing dietary forage did not influence milk yield or composition, although we observed changes in dry matter intake. Time spent chewing, eating, and ruminating (expressed as minutes per day or as minutes per kilogram of NDF intake) were not affected by reducing dietary forage. However, addition of chopped wheat straw to the diets resulted in greater time spent chewing and eating per kilogram of peNDF 1.18 consumed. Reducing dietary forage from 52 to 39% did not affect ruminal pH, ruminal digesta volume and mass, ruminal pool size of NDF or starch, ruminal digesta mat consistency, or microbial N yield. Ruminal acetate-to-propionate ratio was reduced, ruminal turnover rates of NDF and starch were greater, and total-tract digestibility of fiber diminished as dietary forage content decreased. Reducing the dietary forage content from 52 to 39% of dry matter, while increasing wheat straw inclusion to maintain chewing and rumen function, resulted in similar milk yield and composition although feed intake increased. With the lower starch diets in this short-term study, the minimal forage content to maintain lactational performance was between 39 and 43%.

An in vitro perifusion system was developed for bovine hypothalamic tissue to examine the role of alpha 2-adrenergic receptors in the regulation of growth hormone-releasing hormone (GHRH) and somatostatin (SRIF) release. Up to three sagittal slices (600 microns) of hypothalamus, immediately parallel to the midline, were cut in an oxygenated balanced salt solution at 4 degrees C, placed in 5 cc syringes, and perifused at 37 degrees C with oxygenated minimum essential medium-alpha at a flow rate of 0.15 ml/min. Three experiments were conducted, and medium effluent was collected every 20 min before (two samples), during (one or three samples), and after (six samples) treatment. Areas under GHRH and SRIF response curves (AUC), adjusted by covariance for pretreatment values, were calculated from samples collected during the treatment/post-treatment period. Location from which slices were cut, relative to the sagittal midline, had no effect on basal release of GHRH and SRIF, but variation in basal release of GHRH and SRIF differed among animals. Medium containing 60 mM KCI increased AUC for GHRH 39% and 161% for SRIF when compared with perifusion of medium alone, thereby verifying that tissue remained viable for at least 14 hr. Activation of alpha 2-adrenergic receptor with 10(-6) and 10(-4) M clonidine increased AUC for GHRH from 54.8 (control) to 79.1 and 108.7 +/- 2.5 ng.ml-1 min for 10(-6) M and 10(-4) M clonidine, respectively. Guanabenz, another alpha 2-adrenergic receptor agonist, at 10(-8), 10(-6), and 10(-4) M also increased GHRH release from 45.5 (control) to 52.8, 66.2, and 86.7 +/- 1.6 ng.ml-1 min, respectively. Clonidine and guanabenz did not affect release of SRIF. An alpha 2-adrenergic receptor antagonist, idazoxan, blocked clonidine-induced release of GHRH without affecting release of SRIF. We concluded that alpha 2-adrenergic receptor stimulation of in vivo growth hormone secretion in cattle is mediated via an increase in release of GHRH and not a change in release of SRIF.

An in vitro perifusion system for bovine hypothalamic tissue was utilized to examine the role of D1 and D2 dopamine receptors in the regulation of somatostatin (SRIF) and growth hormone-releasing hormone (GHRH) release. Up to three sagittal slices (600 microns) of bovine hypothalamus, immediately parallel to the midline, were cut in an oxygenated balanced salt solution at 4 degrees C, placed in 5 cc syringes, and perifused at 37 degrees C with oxygenated minimum essential medium-alpha at a flow rate of 0.15 ml/min. Five experiments were conducted, and medium effluent was collected every 20 min before (two samples), during (one or three samples), and after (six samples) treatment. Areas under SRIF and GHRH response curves (AUC), adjusted by covariance for pretreatment values, were calculated from samples collected during the treatment/post-treatment period. Activation of D1 receptor with 10(-8) M and 10(-6) M SKF 38393 increased AUC for SRIF from 5.6 (control) to 420 and 500 +/- 57.8 ng.ml-1 min, but 10(-10) M SKF 38393 was ineffective. Relative to controls, release of GHRH was decreased 50% in the 10(-6) M SKF 38393 group. Blockade of D1 receptors with SCH 23390 had no effect on basal release of either SRIF or GHRH, but prevented SKF 38393-induced release of SRIF and SKF 38393-induced suppression of GHRH. In contrast, quinelorane, a D2 receptor agonist, and haloperidol, which blocks D2 receptors, did not affect release of SRIF or GHRH. We concluded that activation of D1 dopamine receptors, but not D2 dopamine receptors, stimulates release of SRIF and inhibits release of GHRH from the bovine hypothalamus.

An in vitro perifusion system for bovine hypothalamic tissue was used to determine if growth hormone-releasing hormone (GHRH) and somatostatin (SRIF) modulate each other's release, and whether SRIF mediates D1-agonist-induced suppression of GHRH in cattle. Up to three sagittal slices (600 microns) of bovine hypothalamus, immediately parallel++ to the midline, were cut in an oxygenated balanced salt solution at 4 degrees C, placed in 5 cc syringe barrels, and perifused at 37 degrees C with oxygenated minimum essential medium-alpha at a flow rate of 0.15 ml/min. Three experiments were conducted, and medium effluent was collected every 20 min before (two samples), during (one or three samples), and after (six samples) treatment. Areas under GHRH and SRIF response curves (AUC), adjusted by covariance for pretreatment values, were calculated from samples collected during the treatment/post-treatment period. Perifusion of SRIF at 10(-6) M and 10(-4) M decreased AUC for GHRH from 86.3 (control) to 65.4 and 59.5 +/- 6.3 ng.ml-1 min, but 10(-8) M SRIF was ineffective. Relative to controls, 10(-8).10(-6), and 10(-4) M GHRH increased release of SRIF 190, 675, and 1,135%, respectively. Activation of D1 receptors with 10(-6) M SKF 38393 increased AUC for SRIF from 12.5 ng.ml-1 min (control) to 484.9 ng.ml-1 min and decreased AUC for GHRH from 36.4 ng.ml-1 min (control) to 18.2 ng.ml-1 min. Blockade of SRIF action with a SRIF antagonist, cyclo-[7-aminoheptanoyl-phe-D-trp-lys-thr(bzl)], increased release of GHRH 1.9-fold. In addition, the SRIF antagonist blocked SKF 38393-induced suppression of GHRH. We concluded that GHRH and SRIF interact within the bovine hypothalamus/pituitary stalk to modulate the release of the other. Moreover, SRIF mediates the inhibitory effects of activation of D1 receptors on release of GHRH in cattle.

Our objective was to study the effects of recombinant bovine growth hormone and bovine growth hormone-releasing factor on gene expression of glucose transporters in the liver and kidney of lactating cows. Primiparous cows received 29 mg/d of bovine growth hormone or 12 mg/d of bovine growth hormone-releasing factor by continuous intravenous infusion or no treatment (control) from 118 to 181 d postpartum. Growth hormone or growth hormone-releasing factor did not influence the mRNA abundance of the liver-type glucose transporter in the liver or kidney. Similarly, neither growth hormone-releasing factor nor growth hormone altered the mRNA abundance of the erythrocyte-type or the intestinal-type glucose transporter in the kidney, but large individual differences were observed in the mRNA abundance of the intestinal-type glucose transporter in liver.

In vitro and in vivo experiments were used to determine the relationship between 5-hydroxytryptaminergic and alpha 2-adrenergic receptors in regulation of growth hormone secretion in cattle. Activation of 5-hydroxytryptaminergic receptors (10(-8), 10(-6), 10(-4) M quipazine) or alpha 2-adrenergic receptors (10(-8), 10(-6), 10(-4) M clonidine) had no effect on secretion of growth hormone from perifused anterior pituitary cells. In vivo, quipazine (0.2 mg/kg body wt, i.v.) and clonidine (8 micrograms/kg body wt, i.v.), when injected separately, each maximized secretion of growth hormone in Holstein steers. However, concurrent administration of quipazine and clonidine at these doses additively increased secretion of growth hormone (mean areas under curves = 439, 914, 1425, and 2359 +/- a pooled SEM of 246 ng.ml-1.min for vehicle, clonidine, quipazine, and quipazine plus clonidine treatments, respectively). Blockade of 5-hydroxytryptaminergic receptors with cyproheptadine (0.2 or 1.0 mg/kg body wt, s.c., 0740 hr) decreased basal concentrations of growth hormone but had no effect on the ability of clonidine (8 micrograms/kg body wt, i.v., 0840 hr) to increase secretion of growth hormone (mean areas under curves = 591, 1218, 363, 1087, and 1002 +/- a pooled SEM of 177 ng.ml-1.min for vehicle-vehicle, vehicle-clonidine, 0.2 mg cyproheptadine-vehicle, 0.2 mg cyproheptadine-clonidine and 1.0 mg cyproheptadine-clonidine treatments, respectively). Blockade of alpha 2-adrenergic receptors with either yohimbine (5 mg/kg body wt, s.c., 0740 hr) or idazoxan (20 mg/kg body wt, s.c., 0740 hr) suppressed both basal and 5-hydroxytryptaminergic receptor-stimulated (0.2 mg quipazine/kg body wt, i.v., 0840 hr) secretion of growth hormone (mean areas under curves = 568, 1252, 410, and 558 +/- a pooled SEM of 108 ng.ml-1.min for vehicle-vehicle, vehicle-quipazine, yohimbine-vehicle, and yohimbine-quipazine treatments, respectively, and means of 553, 1468, 194, and 686 +/- a pooled SEM of 221 ng.ml-1.min for vehicle-vehicle, vehicle-quipazine, idazoxan-vehicle, and idazoxan-quipazine treatments, respectively). We conclude that two mechanisms in the central nervous system mediate 5-hydroxytryptaminergic receptor-stimulated secretion of growth hormone in cattle; one independent and another dependent on alpha 2-adrenergic receptors, possibly via regulation of basal growth hormone secretion. In contrast, alpha 2-adrenergic receptor-induced secretion of growth hormone occurs independently of 5-hydroxytryptaminergic receptors.

Growth hormone-releasing factor is the main endogenous stimulator of somatotropin secretion. Our objective was to compare the effects of recombinant growth hormone-releasing factor and recombinant bST on somatotropin secretion, mammary function, and body composition of lactating, primiparous dairy cows. Cows (118 d of lactation) served as uninfused controls or were infused for 63 d with 12 mg/d of releasing factor or with 29 mg/d of bST. These doses elevated somatotropin in serum to concentrations of similar magnitude. The releasing factor and bST each similarly increased milk yield, yield of milk components, weight of most organs, mobilization of adipose tissue, accretion of lean tissue in the carcass, and metabolic activity (RNA) of mammary tissue. Relative to controls, neither releasing factor nor bST significantly affected cell numbers (DNA) in mammary glands or concentrations of plasmin in milk. None of the variables measured provided evidence for galactopoietic effects of releasing factor independent of somatotropin.

To determine whether bST additively increases milk production in cows milked at different frequencies per day, 118 Holstein primiparous and multiparous cows were milked two or three times daily beginning at parturition and received either 14 mg of bST or no injection beginning at d 75 of lactation. Increased milking frequency from two to three times daily increased 3.5% FCM in multiparous (4.7 kg/d) and primiparous (4.1 kg/d) cows over 305 d. Injection of bST increased FCM 4.3 kg/d in multiparous cows and 5.0 kg/d in primiparous cows over 230 d. Increased milking frequency from two to three times daily reduced milk fat and protein percentages in milk, but bST generally did not affect these percentages. Injection of bST prevented increases in body condition score as lactation advanced, but increasing milking frequency from two to three times daily did not. In general, bST and increased milking frequency additively increased FCM in multiparous and primiparous cows.

Two experiments were conducted to determine the galactopoietic effects of an analog of growth hormone-releasing factor. Specific replacements were made in the AA sequence of growth hormone-releasing factor to produce a growth hormone-releasing factor analog with enhanced stability in vivo. In Experiment 1, 24 Holstein cows were infused i.v. with 0, .33, 1, or 3 mg/d of growth hormone-releasing factor analog for 20 d. Infusion of the analog increased mean serum concentrations of somatotropin, IGF-I, and NEFA. The analog also increased milk yield as much as 44% relative to that of controls. In Experiment 2, 24 Holstein cows were infused i.v. with 0, .11, .33, or 1 mg/d of analog for 60 d. Infusion of analog increased serum concentrations of somatotropin, IGF-I, and NEFA in a dose-dependent manner. At 1 mg/d, the analog increased SCM yield 28% throughout the infusion period relative to the 0 mg/d controls. In summary, a growth hormone-releasing factor analog increased milk yield and serum concentrations of somatotropin and IGF-I. Furthermore, this galactopoietic activity was mediated through the somatotropic axis.

Effects of activation or blockade of alpha 2-adrenergic receptors on serum growth hormone concentrations were studied in Holstein steers (115 +/- 4 days of age; 112 +/- 4 kg body wt). A pelleted diet was available ad libitum for 2 hr each day. Serum growth hormone concentrations were greater for 80 min immediately before feeding compared with 80 min immediately after removal of feed. Relative to saline-injected controls, activation of alpha 2-adrenergic receptors with clonidine (2 micrograms/kg body wt iv) before feeding rapidly increased serum growth hormone concentrations, but clonidine had no effect when administered after feeding. Compared with vehicle-injected controls, blockade of alpha 2-adrenergic receptors with either idazoxan (20 mg/kg body wt sc) or yohimbine (5 mg/kg body wt sc) decreased serum growth hormone concentrations before as well as after feeding. Feeding abolished the stimulatory alpha 2-adrenergic receptor-mediated increase in growth hormone secretion. Our data support the hypothesis that alpha 2-adrenergic receptor stimulation is an essential event mediating pulsatility of growth hormone secretion before feeding and is required to maintain basal concentrations after feeding.

Eight Holsteins per group received 12 mg/d of recombinant growth hormone-releasing factor or 29 mg/d of recombinant bST or served as untreated controls for 60 d. Milk yield and composition were measured for 10 d before infusion, during infusion (d 0 to 59), and for 20 d after infusion ended. Compared with controls, bST and growth hormone-releasing factor increased SCM during infusion. The SCM yield of cows treated with growth hormone-releasing factor was greater than that of bST-treated cows during the final 20 d of infusion. Relative to controls, bST and growth hormone-releasing factor increased serum concentrations of somatotropin and IGF-I during infusion. Concentrations of somatotropin and IGF-I in serum of bST- and growth hormone-releasing factor-treated cows did not differ during infusion. In summary, growth hormone-releasing factor increased SCM yield more than bST, despite similar serum concentrations of somatotropin and IGF-I. Thus, the galactopoietic action of growth hormone-releasing factor was not explained solely by elevation of total radioimmunoassayable somatotropin and IGF-I in serum.

Continuous intravenous infusion of bovine growth hormone-releasing factor (bGRF) increases milk synthesis in dairy cattle by as much as 46%. We have begun to develop a system for delivery and expression of a synthetic bGRF gene in cultured bovine cells using the provirus of the bovine leukemia virus (BLV). The gene encoding synthetic bGRF, constructed from eight overlapping oligonucleotides, was fused to the whey acidic protein promoter (WAP) or the mouse mammary tumor virus promoter (MMTV). These plasmids, termed pWAP.GRF and pMMTV.GRF, were able to induce transcription of bGRF upon transfection into Madin-Darby bovine kidney (MDBK) cells and induction with a lactogenic hormonal milieu (prolactin, hydrocortisone, triiodothyronine, insulin) or dexamethasone. When these constructs were cloned into a BLV vector in place of its oncogenic region, and transfected into MDBK cells, bGRF was expressed. Virus particles were prepared from these cultures and used to deliver the bGRF gene by viral infection into fresh MDBK cells. Northern blot analysis of MDBK total RNA revealed a fivefold higher level of expression of bGRF mRNA in transfected cultures than in virally infected cells, and no expression was detected in control cultures. The bGRF peptide was detected in both cell extracts and media samples from transfected cultures but was not detected in cell extracts or media samples from virally infected cells. This provirus construct may prove useful as a delivery system for peptides into cattle.

The United States Environmental Protection Agency has identified estrogens from animal feeding operations as a major environmental concern, but few data are available to quantify the excretion of estrogenic compounds by dairy cattle. The objectives of this study were to quantify variation in estrogenic activity in feces and urine due to increased dietary inclusion of phytoestrogens. Ten Holstein heifers were assigned to 2 groups balanced for age and days pregnant; groups were randomly assigned to treatment sequence in a 2-period crossover design. Dietary treatments consisted of grass hay or red clover hay, and necessary supplements. Total collection allowed for sampling of feed refusals, feces, and urine during the last 4 d of each period. Feces and urine samples were pooled by heifer and period, and base extracts were analyzed for estrogenic activity (estrogen equivalents) using the yeast estrogen screen bioassay. Feces and urine samples collected from 5 heifers were extracted and analyzed using liquid chromatography-tandem mass spectrometry (LC-MS/MS) to quantify excretion of 7 phytoestrogenic compounds. Excretion of 17-beta estradiol equivalents in urine was higher and tended to be higher in feces for heifers fed red clover hay (84.4 and 120.2 mg/d for feces and urine, respectively) compared with those fed grass hay (57.4 and 35.6 mg/d). Analysis by LC-MS/MS indicated greater fecal excretion of equol, genistein, daidzein, coumestrol, and formononetin by heifers fed red clover hay (1634, 29.9, 96.3, 27.8, and 163 mg/d, respectively) than heifers fed grass hay (340, 3.0, 46.2, 8.8, and 18.3 mg/d, respectively). Diet had no effect on fecal biochanin A or 2-carbethoxy-5, 7-dihydroxy-4'-methoxyisoflavone. Four phytoestrogens were detected in urine (2-carbethoxy-5, 7-dihydroxy-4'-methoxyisoflavone, daidzein, equol, and formononetin) and their excretion was not affected by diet. Identifying sources of variation in estrogenic activity of manure will aid in the development of practices to reduce environmental estrogen accumulation.

The objective of this study was to determine if melatonin fed in the middle of a long day (l6L:8D) reduces mammary parenchymal growth and reduces serum prolactin in prepubertal heifers, thereby mimicking the effects of a short photoperiod. Sixteen prepubertal Holstein heifers were maintained under natural May to August environmental conditions of Michigan plus supplemental lighting to provide a photoperiod of l6L:8D (lights-on 0600-2200 h). At the midpoint of each day, 8 animals were individually fed melatonin (4 mg/100 kg body weight) and 8 were individually fed vehicle (95% ethanol) in 200 g of a grain concentrate mixture. Blood samples were collected at 1-h intervals for 25 h beginning on Day 67. On Day 70 or 72 heifers were slaughtered. No differences were found in body weight gain between melatonin- and vehicle-fed animals. Parenchyma of mammary glands from melatonin-fed heifers had a lower content (24%) and concentration (17%) of deoxyribonucleic acid but a greater concentration of triglyceride (24%) than that of controls. Mean serum prolactin concentration was 27% lower in melatonin-fed animals. In both groups, serum concentrations of prolactin varied throughout the day, with greatest values occurring between 1100 and 1800 h in positive association with changes in ambient temperature. We conclude that melatonin orally administered to prepubertal heifers reduced mammary parenchymal growth and concentration of prolactln in serum. The data support the hypothesis that melatonin mimicks photoperiodic effects on mammary growth and prolactin secretion in cattle.

The effects of elevated circulating concentrations of prolactin were examined on neurochemical estimates of activity of dopaminergic neurons in the infundibulum/pituitary stalk of Holstein bull calves (8-10 weeks of age). Activity of these neurons was estimated by measuring the accumulation of dihydroxyphenylalanine, the immediate precursor of dopamine, 15 min after an intravenous injection of the aromatic L-amino acid decarboxylase inhibitor, 3-hydroxybenzylhydrazine. Subcutaneous injections of the dopamine antagonist haloperidol every 6 hr for 1 day increased serum concentrations of prolactin and accumulation of dihydroxyphenylalanine in the infundibulum/stalk. Intravenous infusions of prolactin for 1 or 9 days increased accumulation of dihydroxyphenylalanine in the infundibulum/stalk, indicating that these neurons remain responsive to elevated prolactin for at least 9 days. It is concluded that elevated concentrations of prolactin in blood stimulate dopaminergic neurons in the infundibulum/pituitary stalk of bull calves. We speculate that these neurons may be analogous to the tuberoinfundibular dopaminergic neurons that regulate prolactin in rats.

The somatotropic axis, consisting of growth hormone (GH), GH receptor (GHR), insulin-like growth factor (IGF)-I, IGF binding proteins (IGFBP), and IGF receptors, controls growth and mammary development in heifers. Manipulation of the axis with recombinant bovine somatotropin (rbST) improves heifer growth and reduces age at first calving. The effects of rbST are influenced by dietary energy through partially understood mechanisms. The objective was to characterize the somatotropic axis in Holstein heifers fed a diet for either low or high rate of gain and treated with or without rbST. Heifers (120 d of age) were assigned to one of 2 diets to gain either 0.8 kg/d (low, n = 18) or 1.2 kg/d (high, n = 20). Within each diet, half of the heifers (n = 9 for low and n = 10 for high) received daily rbST injections (25 microg/kg of body weight). Treatments and diets continued until slaughter (2 mo after puberty). Blood was collected 2x per week, and a frequent sampling window was performed 1 d before slaughter. Liver was collected at slaughter. Feeding a high diet or treating with rbST increased serum IGF-I and decreased serum IGFBP-2. The observed changes in serum IGF-I and IGFBP-2 were reflected in their respective liver mRNA amounts. Feeding a high diet decreased serum GH concentrations after rbST injection, but the stimulatory effect of rbST on serum IGF-I was nonetheless greater in high-diet heifers. The differential IGF-I response may be explained by greater GHR 1A in the liver of high-diet heifers. We conclude that a high-gain diet modifies the somatotropic axis in rbST-treated heifers by decreasing serum GH but increasing serum IGF-I after rbST treatment. Greater IGF-I (indicative of an increased GH response) may be a consequence of greater GHR 1A expression in the liver.

Our objective was to determine if prepubertal rate of body weight (BW) gain, independent of diet, was related to mammary development of dairy heifers. Data from two studies recently conducted at Michigan State University were used to identify factors, within a dietary treatment group, that would account for variation in first lactation milk production or amount of mammary parenchymal DNA at the time of puberty. Factors analyzed for variation in milk production during first lactation were: postpartum BW, prepubertal BW gain, gestational BW gain, postpartum BW gain, body condition score (BCS) at breeding, and BCS at calving. Factors analyzed for variation in mammary parenchymal DNA at puberty were: BW at slaughter, age at puberty, prepubertal BW gain and body protein and body fat content at slaughter. For both analyses, prepubertal BW gain did not account for any of the variation in mammary development. The only significant covariate for the milk production model (r2 = 0.44) was BCS at breeding. Similarly, the only significant covariate in the parenchymal DNA model (r2 = 0.22) was body fat content at slaughter. These results suggest that, within a dietary treatment, heifers that grow faster do not have impaired mammary development, and increased body fatness may be a better predictor of impaired mammary development than BW gain.

The objective was to determine whether increased dietary protein would enhance mammary development in prepubertal heifers fed for rapid body growth (1.2 kg/d). Fifty-four Holstein heifers (weighing approximately 134 kg) were assigned to one of three treatments. Heifers were fed a total mixed ration with metabolizable energy at 2.85 Mcal/kg and metabolizable protein at low, standard, or high concentrations (37, 41, or 44 g/Mcal of metabolizable energy, respectively) from 3.5 mo of age until slaughter at approximately 46 d after puberty. Heifers fed low, standard, and high protein gained 1130, 1170, and 1180 g/d, respectively. Dietary protein did not affect age or weight of heifers at puberty or slaughter, withers height gain, or carcass composition. Average mammary parenchymal DNA content for heifers on diets of low, standard, and high protein was 595, 619, and 670 mg/100 kg of body weight, respectively, and was not significantly different. However, for heifers that attained puberty early, those fed low protein had 33% less parenchymal DNA than those fed high protein, even though their body growth and carcass composition were not compromised. We conclude that dietary protein does not have a major effect on mammary development of rapidly grown prepubertal heifers, provided the diet contains adequate protein for normal body growth. But we suggest that feeding low-protein diets increases the risk of impaired mammary development when heifers are fed for rapid growth and attain puberty early and that the new National Research Council guidelines for protein relative to energy seem adequate for optimal mammary development.

The purpose of this experiment was to determine the role of growth hormone-releasing hormone (GHRH) and somatostatin (SRIH) neurons in mediating α2-adrenergic receptor-induced stimulation of growth hormone (GH) secretion in cattle. Our first objective was to determine if stimulation of α2-adrenergic receptors increases activity of GHRH neurons in the arcuate nucleus (ARC) and/or decreases activity of SRIH neurons in periventricular (PeVN) and ARC nuclei. Clonidine (an α2-adrenergic agonist) or vehicle (saline) were injected i.v. into steers and dual-label immunohistochemistry was performed to quantify the number of GHRH and SRIH neurons expressing Fos and Fos-related antigens (Fos/FRA) as markers of neuronal activity. Clonidine increased concentrations of GH in serum and decreased activity of SRIH neurons in the PeVN, but not in the ARC. Clonidine did not alter activity of GHRH neurons in the ARC. Our second objective was to determine if clonidine decreases secretion of SRIH from perifused slices of hypothalami, which contain perikarya and terminals of GHRH and SRIH neurons, and from explants of hypophysial stalk alone, which contain only terminals of GHRH and SRIH neurons. Clonidine failed to alter release of GHRH or SRIH from hypothalamic slices, but stimulated release of GHRH from explants of hypophysial stalk. Blockade of SRIH receptors enabled clonidine to stimulate release of GHRH from slices of hypothalami, but also stimulated release of SRIH. These results suggest that α2-adrenergic-induced secretion of GH occurs via a dual mechanism involving inhibition of SRIH neurons in the PeVN and direct stimulation of GHRH release from axon terminals in the median eminence.

Since the initial report in 1978 of galactopoietic effects of a photoperiod of 16 h of light:8 h of darkness, numerous studies have confirmed long-day stimulation of milk yield. The endocrine factor(s) responsible for increased milk yield, however, has eluded identification. Recent studies suggest that insulin-like growth factor-I (IGF-I) may mediate the galactopoietic response to long day photoperiod. Indeed, long days increase IGF-I in heifers and lactating cows; in the latter case, the response preceded an increase in milk yield. In heifers and cows, the increase in IGF-I is independent of changes in circulating growth hormone. Melatonin feeding to mimic a short-day photoperiod suppressed the increase of IGF-I in heifers induced by long days. However, melatonin feeding had no effect on milk yield in cows. Despite lack of resolution of the endocrine mechanism, dairy producers are interested in how photoperiod management can be integrated with current practices throughout the lactation cycle. There is strong evidence that milk yield responses to long days persist through an entire lactation. Also, long days can be combined with bovine somatotropin (bST) to produce additive increases in milk yield. During the dry period, long days increase the periparturient surge of prolactin. However, relative to long days, short-day treatment during the dry period produces the largest magnitude of response in milk yield during the subsequent lactation. The response to short days during the dry period may be due to a priming effect on the photoperiodic response system. In summary, IGF-I has emerged as a possible mediator of the increase of milk yield in response to long-day photoperiod. Photoperiod can be combined effectively with other management techniques such as bST. Consideration of photoperiod management during the dry period is essential to maximize responses during the subsequent lactation.

To determine effects of rapid prepubertal growth on first-lactation milk production, Holstein heifers were randomly assigned to one of three treatments. Thirty-five heifers were fed a standard diet to meet NRC recommendations and produce 0.8kg of body weight (BW) gain/d (standard). Thirty-five heifers were fed a diet with higher energy (2.8 Mcal of metabolizable energy/kg) and protein (19.7% crude protein; high diet) to produce 1.2kg of BW gain/d (high). Thirty-five heifers were fed the high diet and injected daily with bovine somatotropin (bST) (25µg/kg of BW; high-bST). Diets were fed and bST was injected from 135kg of BW until pregnancy was confirmed. Heifers were inseminated after BW exceeded 363kg. Pregnant heifers were commingled and fed similar diets through gestation, parturition, and lactation. High and high-bST heifers had greater prebreeding average BW gains than standard heifers. Conversely, standard heifers had a greater average BW gain during gestation than high and high-bST heifers. High and high-bST heifers were approximately 90 d younger than standard heifers at first insemination and first parturition. Postpartum BW, body condition scores, and withers heights at parturition, and calving ease scores were not different among treatments. Standard heifers produced 14% more milk than high heifers but not more than high-bST heifers. The high-protein, high-energy diet decreased age at first parturition and first-lactation milk production, but did not affect reproduction. Injection of bST during the pre-pubertal growth period combined with the high diet decreased age at first parturition without reducing milk production.

Secretion of growth hormone (GH) is reduced for several hours after feeding when access to feed is restricted to a 2-hr period each day. We hypothesized that increased secretion of insulin after feeding inhibits release of GH from the anterior pituitary gland. Our objectives were to determine whether: 1) alloxan prevents concentrations of insulin from increasing after feeding steers; 2) concentrations of GH remain high after feeding alloxan-treated steers; and 3) GH-releasing hormone (GHRH) stimulates greater release of GH in alloxan-treated, than in control, steers after feeding. Steers were injected iv with either saline (control) or with alloxan (110 mg/kg) (n = 4 per group). Concentrations of insulin were not different (P = 0.61) between control and alloxan-treated steers before feeding (87.5 ± 33.6 pmol/l). However, alloxan prevented insulin from increasing (P < 0.001) after feeding (131.8 pmol/l) compared with control steers (442.0 pmol/l) (pooled SEM = 47.5). Overall, GH was higher (P < 0.05) in alloxan-treated (6.4 ng/ml) than in control steers (3.7 ng/ml) (pooled SEM = 0.7), but GH decreased (P < 0.001) after feeding in both groups. Iv injection of GHRH stimulated release of GH 1 hr before, but not when injected 1 hr after feeding (P < 0.001). In addition, net areas under the GH curve were not significantly different between control and alloxan-treated groups. We conclude that increased concentrations of insulin after feeding do not mediate feeding-induced suppression of GH secretion in steers.

The mRNA for growth hormone receptor is transcribed from at least three different promoters in cattle. The first promoter (P1) is liver-specific and transcribes growth hormone receptor mRNA containing exon 1A (growth hormone receptor 1A). The second and third promoters (P2 and P3) are active in a variety of tissues and transcribe growth hormone receptor mRNA containing exon 1B and 1C. The objective was to characterize P1 activity by measuring the amount of growth hormone receptor 1A mRNA in liver of dairy cows at different stages of lactation as well as after administration of recombinant bovine somatotropin (rbST). In study 1, liver RNA was isolated from Holstein cows during the dry period (nonlactating, n = 6) and during early (n = 6), mid (n = 6), and late (n = 11) stages of lactation. Six of the late-lactation cows received injections of rbST (25 mg/d) for 7 d prior to collection of liver tissue. In study 2, lactating Holstein cows received either no infusion (control, n = 10) or continuous infusion of rbST (29 mg/d, n = 10) for 63 d. The amount of growth hormone receptor 1A mRNA was decreased in early- and mid-lactation cows compared with late-lactation cows or nonlactating cows (study 1). Administration of rbST increased growth hormone receptor 1A mRNA (studies 1 and 2). The total amount of growth hormone receptor transcribed from alternative promoters (growth hormone receptor P2 and P3) remained unchanged during different stages of lactation or in response to rbST. We conclude that changes in liver growth hormone receptor mRNA in lactating dairy cattle primarily depend on growth hormone receptor P1 activity.

Forty Holstein heifers [body weight (BW) = 126 kg] were blocked by BW into groups of 4, and, within each block, heifers were randomly assigned to one of four treatments. Twenty heifers had ad libitum access to a diet formulated to produce a BW gain of 0.8 kg/d (control diet), and 20 heifers had ad libitum access to a diet formulated to produce a BW gain of 1.2 kg/d. (high diet). Half of the heifers fed each diet were injected daily with bovine somatotropin (bST; 25 micrograms/ kg of BW). The high diet increased daily BW gain as well as body condition score. Injection of bST also increased daily BW gain, but did not affect body condition score. The high diet reduced age at puberty by 58 d, but did not affect BW, withers height at puberty, or pelvic area at slaughter. Injection of bST had no effect on age at puberty, but increased BW, withers height at puberty, and pelvic area at slaughter. The high diet did not affect mammary parenchymal DNA, RNA, or the ratio of RNA to DNA. The injection of bST increased mammary parenchymal DNA, RNA, and the ratio of RNA to DNA. The high diet was more cost effective for rearing dairy heifers from 120 d of age to potential breeding size (> or = 363 kg of BW and postpubertal) than was the control diet. In conclusion, the high protein, high energy diet increased growth rate without detrimental effects on mammary development. Injection of bST increased BW, skeletal size, and mammary development.

Lactating, primiparous Holstein cows were continuously infused for 63 d with either recombinant growth hormone-releasing factor or recombinant bST or were not infused (control). The releasing factor and bST treatments reduced fat synthesis from acetate and lipoprotein lipase activity in omental adipose tissue. Also, both treatments increased hormone-sensitive lipase activity per gram of adipose tissue and reduced fatty acid esterification per milligram of protein in adipose tissue. Protein concentration in adipose tissue with both treatments was greater than that of the control, although the protein concentration was less in adipose tissue from cows treated with the releasing factor than from those treated with bST. In mammary tissue, releasing factor increased fat synthesis from acetate more than did bST treatment, but percentages of fat or short-chain fatty acids in milk were not different between treatments, nor were differences significant between treatments and control for fatty acid esterification or lipoprotein lipase activity in mammary tissue or gluconeogenesis and fatty acid esterification in liver. We conclude that protein concentration was lower in omental adipose tissue of cows treated with releasing factor than that in tissue of cows treated with bST, although both treatments similarly suppressed synthesis of fat from acetate and activity of lipoprotein lipase.

Our objective was to compare the effects of recombinant bovine growth hormone-releasing factor and recombinant bST on the somatotropic cascade in lactating dairy cows. Primiparous cows were killed after 63 d of continuous daily infusion with 12 mg of releasing factor or 29 mg of bST or no infusion (controls). Both hormone infusions similarly increased mean concentration of somatotropin in serum, but pulsatility of somatotropin in serum was greater for cows given releasing factor. Both hormone infusions increased the amounts of IGF-I in serum, IGF-I mRNA in liver, and IGF-binding protein-3 in serum and decreased IGF binding protein-2 in serum, but these effects were less for cows given releasing factor than for those given bST. Both infusions decreased the number of free binding sites for IGF-I in mammary tissue. In liver, treatment did not alter the abundance of mRNA for the somatotropin receptor or the number of free binding sites for somatotropin. Results suggest that endogenous somatotropin is less effective as an IGF-I secretagogue than is exogenous bST infused continuously, yet the releasing factor and bST increased milk yield similarly. We conclude that growth hormone-releasing factor stimulates milk synthesis mostly through the same mechanisms as bST, but that serum IGF-I alone is not a good indicator of the galactopoietic potency of the two hormones.

Effects of activation or blockade of 5-hydroxytryptaminergic receptors on concentrations of growth hormone and somatostatin in serum were studied in Holstein steers (mean +/- SEM: 159 +/- 8 days of age; 160 +/- 9 kg of body wt). A pelleted diet was available ad libitum between 1000 and 1200 hr each day. Blood was sampled from a cannula in a jugular vein. Peak concentration of growth hormone in serum within 1 hr before feeding (12.8 +/- 3.8 ng/ml) was greater than peak concentration within 1 hr after removal of feed (3.3 +/- 1.0 ng/ml). In contrast, concentration of somatostatin in plasma did not change from 1 hr before feeding through 1 hr after removal of feed (47.3 to 43.0 +/- 4.5 pg/ml). Relative to saline-injected controls, activation of 5-hydroxytryptaminergic receptors with quipazine (0.5, 0.1, and 0.2 mg/kg body wt, iv) increased the area under growth hormone response curves 5.5- to 25-fold before and after feeding. In another experiment, injection of 0.1 mg of quipazine/kg body wt at 1300 hr increased the concentration of growth hormone in serum 7.8-fold compared with controls, but had no effect on concentration of somatostatin in plasma. Relative to water-injected controls, blockade of 5-hydroxytryptaminergic receptors with cyproheptadine (0.2 mg/kg body wt, sc) decreased the area under growth hormone response curves for at least 110 min before feeding (71%) and after removal of feed (69%). The data support the hypothesis that 5-hydroxytryptaminergic receptors are involved in stimulation of pulsatile growth hormone secretion in meal-fed cattle. Lack of a change in concentration of somatostatin in plasma with respect to time of meal-feeding or after injection of the 5-hydroxytryptaminergic-receptor agonist quipazine suggests that 5-hydroxytryptaminergic receptor-stimulated growth hormone secretion is likely mediated within the central nervous system, rather than by meal-induced changes in peripheral secretion of somatostatin.

Forty-one Holstein cows were injected with 0, 5, or 14 mg/d of bST for the last 46 +/- 6 d before parturition. Compared with data for controls, the 5- and 14-mg doses of bST increased apparent protein synthesis about 16% before parturition. Exogenous bST before parturition increased apparent protein degradation 30% during wk 1 after parturition. During wk 1 of lactation, 14 mg of bST also increased milk protein yield 33%. No treatment differences were present in concentration of serum NEFA, body condition score, or thickness of subcutaneous fat. Therefore, administration of bST before parturition did not alter metabolism of subcutaneous fat. Prepartum treatment with 5 and 14 mg of bST increased and maintained serum somatotropin at 6.5 and 22.7 ng/ml, respectively, compared with 1.6 ng/ml in controls. Concentrations of serum IGF-I were initially increased but were not maintained as parturition approached. On d -23, IGF binding protein 3 was increased 65% but was not different among groups by d -7. For groups administered the 5 and 14 mg/d of bST, IGF binding protein 2 was decreased 40%. Administration of bST before parturition increased protein reserves and stimulated milk protein yield for 1 wk but did not alter metabolism of subcutaneous fat. Furthermore, energy balance appeared to be a major regulator of concentrations of IGF binding protein 3 and responsiveness of IGF-I to exogenous somatotropin before parturition.

Static primary cultures of bovine anterior pituitary (AP) cells were utilized to study the effect of sex steroids on basal growth hormone (GH) and GH-releasing hormone (GRF)-stimulated release of GH. The AP cells (5 x 10(5) cells/well) were allowed to attach for 72 hr and become confluent before treatments were imposed. Cells were incubated for an additional 24, 48 or 72 hr with either estradiol-17 beta (E2, 10(-11) to 10(-8) M), testosterone (T, 10(-8) to 10(-5) M), dihydrotestosterone (DHT, 10(-9) to 10(-6) M) or 5 alpha-androstane-3 alpha, 17 beta-diol (3 alpha-diol, 10(-11) to 10(-8) M). Media were collected every 24 hr and GH concentrations determined by RIA. Incubation of calf AP cells with gonadal steroids did not affect (P0.05) basal GH released at 24, 48, or 72 hr. In another experiment, calf AP cells were incubated with the same concentrations of the steroids for 24 hr, media harvested, cells washed and challenged in serum-free media for 1 hr with bovine GRF 1-44-NH2 (10(-8) M). In non-steroid treated wells, GRF increased (P0.05) GH from 58 to 134 ng/ml. Incubation with E2 or 3 alpha-diol did not affect (P0.05) GRF-induced GH release; however, preincubation with T (10(-5) M) and DHT (10(-9), 10(-8) and 10(-7) M) increased (P0.05) GRF-induced GH release above control concentrations (195, 235, 190 and 185 ng/ml, respectively). At the doses tested, sex steroids did not affect basal release of GH, but androgens increased responsiveness of somatotropes to GRF.

Holstein cows received 12mg/d of growth hormone-releasing factor (continuous i.v. infusion, n=5), 14mg/d of bST (single daily i.m. injection, n=8), or no treatment (controls, n=8) for 60 d. Compared with controls (31.6kg/d), bST and growth hormone-releasing factor increased milk yield to 34.2 and 37.0kg/d, respectively. The increase in milk yield induced by the growth hormone-releasing factor was greater than that for bST. Milk yield was not different among groups following cessation of treatment. Milk energy output was 24.2Mcal/d in controls, and growth hormone-releasing factor increased milk energy output to 28.5Mcal/d. Milk energy output of cows receiving bST was 26.1Mcal/d. Growth hormone-releasing factor increased DMI (23.2kg/d) over that of controls (21.1kg/d), whereas bST (21.5kg/d) did not. Relative to controls, bST increased averaged daily serum somatotropin from 1.3 to 7.6ng/ml and insulin-like growth factor-I from 67.5 to 116.0ng/ml. Relative to bST, growth hormone-releasing factor increased serum somatotropin to 16.3ng/ml and insulin-like growth factor-I to 202.6ng/ml. Relative to control (115.8meq/dl) and bST (158.1meq/dl), growth hormone-releasing factor increased plasma NEFA (230.3meq/dl). During treatment, calculated energy balance was negative for cows receiving growth hormone-releasing factor but positive for bST and control cows. Milk composition, body condition score, BW, and apparent digestibility of DM were not different among treatments. We conclude that i.v. infusion of 12mg/d mg of growth hormone-releasing factor has greater galactopoietic activity than i.m. injections of 14mg/d of bST. These data support the concept that the galactopoietic effects of growth hormone-releasing factor are mediated via increased secretion of somatotropin and insulin-like growth factor-I.

The effects of ambient temperature on serum concentrations of prolactin and neurochemical estimates of activity of dopaminergic neurons projecting to the infundibulum/pituitary stalk were investigated in Holstein bull calves. Accumulation of 3,4-dihydroxyphenylalanine (DOPA) in the infundibulum/pituitary stalk after intravenous injection of a decarboxylase inhibitor was used to estimate activity of these dopaminergic neurons. Increasing ambient temperature from 21 to 33 degrees C for 22 hr increased serum concentrations of prolactin and decreased activity of the dopaminergic neurons. Conversely, reducing ambient temperature from 22 degrees C to 11 degrees C decreased serum concentrations of prolactin and increased activity of these dopaminergic neurons. It is suggested that alterations in activity of dopaminergic neurons terminating in the infundibulum/pituitary stalk of bull calves may mediate acute temperature-induced changes in secretion of prolactin.

Twenty-four Holstein cows were infused intravenously with sterile water (placebo) or 1, 3, or 12 mg of recombinant bovine somatotropin-releasing factor 1-45-homoserine lactone/d for 60 d. Relative to placebo (22.8 kg/d), 3 and 12 mg of somatotropin-releasing factor increased yield of milk to 28.8 and 33.3 kg/d during infusion. At 1 mg of somatotropin-releasing factor, milk averaged 27.5 kg/d during infusion but was increased above placebo only through 39 d. After infusion of 12 mg somatotropin-releasing factor ended, milk (26.4 kg/d) remained above placebo amounts (20.6 kg/d) for 15 d. Three and 12 mg of somatotropin-releasing factor increased serum somatotropin from .7 (placebo) to 8.2 and 10.3 mg/ml when averaged across 1, 30, and 59 d, whereas 1 mg increased somatotropin to 5.8 ng/ml after 1 d but had no effect at 30 or 59 d. Within 17 h of cessation of somatotropin-releasing factor infusion, serum concentrations of somatotropin were similar across all groups. On d 59, 3 and 12 mg infusions increased insulin-like growth factor I from 115.8 (placebo) to 204.7 and 261.4 ng/ml of serum. We conclude that somatotropin-releasing factor increased serum concentrations of somatotropin and milk yield in a dose-dependent manner for at least 60 d. Also, galactopoietic effects of somatotropin-releasing factor persisted for 15 d independent of increased concentrations of somatotropin in serum following withdrawal of somatotropin-releasing factor.

PRL involvement in periparturient mammary development and onset of milk secretion was studied in 17 multiparous, monotocous Holstein cows. Mammary glands were obtained 10 days prepartum from untreated cows and 10 days postpartum from untreated cows and cows treated with CB154 (2-Br-α-ergokryptin) or CB154 plus PRL. CB154 was administered for 12 days before expected parturition through 10 days postpartum. PRL was infused continuously for 6 days immediately before parturition in cows receiving CB154 plus PRL. Treatment with CB154 reduced basal serum PRL concentrations approximately 80% and blocked the normal surges in serum PRL concentrations at parturition and during milking. Average milk production in cows given CB154 alone was 11.4 kg/day lower than in controls. Periparturient infusion of PRL in cows treated with CB154 (CB154 plus PRL) prevented reductions in milk production. Treatment with CB154 had no effect on feed intake or periparturient serum concentrations of GH, progesterone, and glucocorticoids....

The objectives were to determine if photoperiods of 8 h light:16 h dark (8L:16D) and 16L:8D affect time of day when preovulatory surges of luteinizing hormone (LH) and follicle stimulating hormone (FSH) occur and if photoperiods affect diestrus and estrous concentrations of LH, FSH and prolactin (PRL). Postpubertal Holstein heifers were assigned to receive either 8L:16D (n - 9) or 16L:8D (n = 9) from September 21 through January 31. Hormone concentrations were determined in serum collected at 2-h intervals during diestrus and estrus of a single estrous cycle in autumn and again 4 mo later, during a single estrous cycle in winter. Neither photoperiod nor season affected time of day at which preovulatory LH and FSH surges occurred in heifers. The LH surges did not differ in amplitude and duration within and between photoperiods and within individual animals. In 29 of 33 surges, peak concentrations of FSH coincided with peaks of LH. Peak concentrations of FSH were greater during the winter than during autumn in heifers exposed to 8L:16D, but were not different between autumn and winter in heifers exposed to 16L:8D. Between autumn and winter, surges of FSH were similar in magnitude and duration within individual animals. In comparison with 8L:16D, PRL was increased in heifers that received 16L:8D in autumn, but photoperiod had no effect on PRL during winter. Concentrations of PRL were increased at estrus relative to diestrus in autumn and winter. It is concluded that photoperiods of 8 and 16 h do not affect the timing, amplitude or pattern of preovulatory surges of LH or FSH, the rise in PRL associated with estrus, or length of estrous cycles in postpubertal Holstein heifers.

Milk production and dry matter intake of 21 cows subjected to 16 h of fluorescent light and 8 h dark and of 21 cows subjected to natural light 9 to 12 h daily between October 25 and March 14 were measured beginning in early (37 to 74 days postpartum) and late (94 to 204 days postpartum) lactation. Cows that received 16 h of fluorescent light produced 6.7% (1.4 kg) more milk per day (adjusted for parity and pretreatment production) than cows exposed to natural photoperiods. Increases of milk production with 16 h of fluorescent light were similar for early and late lactation. Photoperiod did not alter percent of fat in milk. Dry matter intake increased 6.1% for cows in 16 h of light, and this increase could account for increased milk yields. Basal prolactin in serum and that released by thyrotropin releasing hormone were 1.5 to 1.8 times greater for cows exposed to 16 h of light than for cows in 9 to 12 h of natural light daily. Photoperiod did not affect release of prolactin by milking. Cold ambient temperatures reduced basal prolactin and prolactin released by thyrotropin releasing hormone but had no effect on concentrations of growth hormone or glucocorticoids. Compared with cows in late lactation, cows in early lactation released 2.4 times more prolactin after milking, but they released similar amounts of prolactin after thyrotropin releasing hormone. Photoperiod did not affect concentrations of growth hormone or glucocorticoids in blood sera.