Identification of differentially expressed transcripts in bovine rumen and abomasum using a differential display method.

The rumen has several important physiological functions: absorption, transport, metabolic activity, and protection. To clarify the molecular basis underlying the physiological function of the rumen, reticulum, omasum, and abomasum, we used mRNA differential display to isolate and identify differentially expressed genes in these tissues. We isolated 18 transcripts that coexpressed in the rumen, reticulum, and omasum. Five genes, ribosomal protein 19 (RPS19), basic helix-loop-helix domain containing class B2 (BHLHB2), NADH dehydrogenase flavoprotein 2 (NDUFV2), exosome component 9 (EXOSC9), and ribosomal protein 23 (RPS23), were highly expressed in the rumen of adult Holstein and Japanese Black cattle. Significant differences of expression were observed in the abomasum compared with the rumen, reticulum, and omasum. To investigate the expression pattern of these genes during the neonatal growth stage, the relative levels of gene expression were analyzed in the rumen and abomasum of 3-wk-, 13-wk-, and 18- to 20-mo-old Holstein cattle. The expression level of RPS19 did not change with age in the rumen and abomasum. The levels of BHLHB2, NDUFV2, and EXOSC9 mRNA in the abomasum decreased (P < 0.05) after weaning and declined (P < 0.05) further in adults; in contrast, expression in the rumen was not altered. Interestingly, the levels of RPS23 mRNA in the rumen increased (P < 0.05) after weaning and further increased in the adult; however, the level of expression of this gene decreased (P < 0.05) in the abomasum with weaning and age. This indicates that the 4 tissues, especially the rumen and abomasum, have different developmental pathways after birth and subsequent onset of rumination.

Effect of conjugated linoleic acid type, treatment period, and dosage on differentiation of 3T3 cells.

This study was conducted to determine effect of CLA and linoleic acid (LA) on cell differentiation, cellular glycerol-3-phosphate dehydrogenase (GPDH) activity, and FA accumulation in differentiating 3T3-L1 cells (3 isomers x 3 treatment periods x 4 doses). The cells were cultured in 24-well plates for proliferation until confluence. Then they were treated with media containing 0, 10, 35, or 70 mg/L (0, 35, 125, or 250 mmol/L, respectively) of LA, cis9,trans11- or trans10,cis12-CLA during early (day 0-2), intermediate and late (day 3-8), or overall (day 0-8) differentiation periods. Dexamethasone, methyl-isobutylxanthine, and insulin were supplemented to the media only for the early period to induce the differentiation. On day 8 of postconfluence the cells were harvested for Oil Red O staining, analysis of GPDH activity, and determination of the FA Concentration. Cellular LA or CLA was found to accumulate in a dose-response manner, mainly during the intermediate/late period. Treatment with trans10,cis12-CLA lowered (P < 0.05) GPDH activity and the concentration of FA including palmitic acid (16:0) and palmitoleic acid (16:1), especially during the intermediate/late and overall periods, or whenever a high dose of 70 mg/L was applied. This also resulted in a higher (P < 0.05) ratio of saturated FA to monounsaturated FA. Treatment with LA or cis9,trans11-CLA lowered cellular FA only when they applied during the early period at a dose of 70 mg/L. The results demonstrated that the inhibitory effects of CLA on differentiation, GPDH activity, and FA accumulation of 3T3-L1 cells are dependent on the isomer type, treatment period, and dose.

Effects of dietary protein and growth hormone-releasing peptide (GHRP-2) on plasma IGF-1 and IGFBPs in Holstein steers.

This study was conduct to determine the influence of dietary protein on the response of plasma insulin-like growth factor-1 (IGF-1) and insulin-like growth factor binding proteins (IGFBPs) to exogenous growth hormone releasing peptide-2 (GHRP-2 or KP 102) in Holstein steers. Eight 16-month-old Holstein steers were grouped by liveweight to two feeding treatments; high protein (HP; CP 1.38 kg/day and TDN 4.5 kg/day DM intake, n=4) or low protein (LP; CP 0.66 kg/day and TDN 4.42 kg/day DM intake, n=4). The experiment was a single reverse design whereby each group was injected twice daily with GHRP-2 (12.5 microg/kg body weight (BW)/day) or saline solution into the jugular vein for a 6-day period. Plasma IGF-1 in the HP group were higher than in the LP group (P<0.05), but plasma 34 kDa IGFBP-2 was lower in the HP than the LP group (P<0.05). The amplitude of the maximum growth hormone (GH) peaks responding to GHRP-2 injection were higher at day 1 than at day 6 of saline or GHRP-2 treatment in both LP and HP steers (P<0.05). The area under the GH response curve for 180 min after the GHRP-2 injection was not significantly different between the LP and the HP groups at days 1 and 6. A response in plasma IGF-1 concentration to GHRP-2 treatment in the HP group was observed at day 1 (198.9+/-18.1 ng/ml), day 2 (195.2+/-21.1 ng/ml) and day 6 (201.3+/-14.8 ng/ml) (P<0.05). No increase in plasma IGF-1 was observed from GHRP-2 administration in the LP group. Although the response of plasma IGF-1 concentration to GHRP-2 administration was increased in the HP group (P<0.05), there was no apparent effect of GHRP-2 treatment on plasma 38-43 kDa IGFBP-3 and 34 kDa IGFBP-2 at days 2 and 6 of treatment. In conclusion, it is proposed that the 34 kDa IGFBP-2 is sensitive to dietary protein level and may play an important role in the regulation of circulating IGF-1 in ruminant. In addition, increased plasma IGF-1 concentration observed in the HP group in response to the GHRP-2 treatment did not appear to affect plasma IGFBPs.

Effects of the administration of growth hormone-releasing peptide-2 (GHRP-2) orally by gavage and in feed on growth hormone release in swine.

The experiments were conducted to determine the effects of the administration of growth hormone-releasing peptide-2 (GHRP-2, also named KP102), both orally by gavage and in feed, on the release of growth hormone (GH) in swine and to investigate whether attenuation of the GH response occurs after short-term treatment with the peptide in feed. In the first experiment, saline or GHRP-2 at doses of 1, 4.5 and 9 mg/kg body weight (BW) was dissolved in 15 ml saline and administered orally as a bolus by gavage to cross-bred castrated male swine (n = 6). Orally administered GHRP-2 stimulated dose-related increases in peak concentrations of GH, with a return to basal by 120 min. After administering GHRP-2 orally, peak concentrations of GH and areas under the GH response curves (GH AUCs) for 180 min were higher (P < 0.05) than those in saline controls. In Experiment 2, GHRP-2 at doses of 0 (served as control), 1, 4.5 and 9 mg/kg BW was mixed in 150 g of feed and offered to cross-bred castrated male swine (n = 6) at 0900 hr and 1700 hr daily for a 3-d period. Administration of 1 mg/kg BW GHRP-2 to swine in feed failed to stimulate the release of GH, but GHRP-2 at doses of 4.5 and 9 mg/kg BW significantly (P < 0.05) increased plasma concentrations of GH after initial and final treatments at 0900 hr on Days 1 and 3 of treatment, respectively. Peak concentrations of GH and GH AUCs for 180 min after the initial and final treatments in the 4.5 and 9 mg/kg BW GHRP-2-treated swine were higher (P < 0.05) than those in controls. After 3 d of treatment with GHRP-2 in feed at doses of 4.5 and 9 mg/kg BW, GH responses to the peptide were maintained. The results of the present study indicate that the administration of GHRP-2 orally by gavage and in feed stimulates the release of GH in swine, and that the GH-releasing effect of the peptide does not become desensitized after short-term administration in feed.

The effects of growth hormone-releasing peptide-2 (GHRP-2) on the release of growth hormone and growth performance in swine.

The effects of GHRP-2 (also named KP102), a new growth hormone-releasing peptide, on the release of growth hormone (GH) and growth performance were examined in swine. The single intravenous (i. v.) injection of GHRP-2 at doses of 2, 10, 30 and 100 microg/kg body weight (BW) to cross-bred castrated male swine stimulated GH release in a dose-dependent manner, with a return to the baseline by 120 min. The peak GH concentrations and GH areas under the response curves (GH AUCs) for 180 min after the injections of GHRP-2 were higher (P < 0.05) than those after the injection of saline. The GH responses to repeated i.v. injections of GHRP-2 (30 microg/kg BW) at 2-h intervals for 6 h were decreased after each injection. The chronic subcutaneous (s.c.) administration of GHRP-2 (30 microg/kg BW) once daily for 30 days consistently stimulated GH release. The GH AUCs for 300 min after the injections on d 1, 10 and 30 of treatment in GHRP-2-treated swine were higher than those in saline-treated swine. However, chronic administration of GHRP-2 caused a partial attenuation of GH response between d 1 and 10 of treatment. The chronic s.c. administration of GHRP-2 also increased average daily gain for the entire treatment period by 22.35% (P < 0.05) and feed efficiency (feed/gain) by 20.64% (P < 0.01) over the saline control values, but did not significantly affect daily feed intake. These results indicate that GHRP-2 stimulates GH release and enhancing growth performance in swine.

The effect of growth hormone-releasing peptide-2 (KP102) administration on plasma insulin-like growth factor (IGF)-1 and IGF-binding proteins in Holstein steers on different planes of nutrition.

This study was conducted to investigate the nutrition-dependent changes in insulin-like growth factor (IGF)-1 and IGF-binding proteins (IGFBPs) with growth hormone releasing peptide-2 (D-Ala-D-betaNal-Ala-Trp-D-Phe-Lys-NH(2); GHRP-2 or KP102) treatment in growing Holstein steers. Eight 13 month-old Holstein steers were grouped on two levels of feed intake (high intake (HI); 2.43% body weight or low intake (LI); 1.22%) and each group was daily injected with KP102 (12.5 microg/kg body weight/day) or saline solution into the jugular vein during 6-day period. The concentration of plasma GH showed an increase after an i.v. bolus injection of KP102 on Day 1 and Day 6 in both the LI and HI groups. Plasma IGF-1 began to increase 10 hr following an i.v. bolus injection of KP102, but this was only observed in the HI group (P < 0.05). Also, the plasma IGF-1 in the HI group with daily injections was significantly greater than the LI group from Day 1 of KP102 administration (P < 0.05). It reached maximum values of 125.1 +/- 7.6 ng/ml after Day 2, and returned to pre-injection levels after Day 4, however, no change in plasma IGF-1 was observed in LI with administration of KP102. During 6 days of treatment, plasma 38-43 kDa IGFBP-3 and 24 kDa IGFBP-4 were significantly higher in KP102 treated steers but only in the HI group (P < 0.05). Plasma 34 kDa IGFBP-2 decreased in the HI group and did not show any change following an injection of KP102. In conclusion, the effect of stimulated endogenous GH with KP102 administration increased plasma IGF-1, 38-43 kDa IGFBP-3 and 24 kDa IGFBP-4 levels in the HI group of growing Holstein steers, but not in the LI one. Thus, we strongly believe that the plasma IGF-1 and IGFBPs response to KP102 treatment is modulated by the nutritional status of growing Holstein steers and the increased plasma IGF-1 concentration with KP102 treatment may be regulated by plasma 38-43 kDa IGFBP-3 and 24 kDa IGFBP-4 in Holstein steers.

Effect of cholinergic blockade on inhibited GH secretion by feeding and intraruminal SCFA infusion in sheep.

The effect of cholinergic blockade on suppressed growth hormone (GH) secretion caused by feeding or the intraruminal infusion of an acetate, propionate, and butyrate mixture (107 and 214 mumol.kg-1.min-1 over 6 h) was examined in ovariectomized ewes. Intraruminal infusion at the rate of 107 mumol.kg-1.min-1 increased peripheral plasma short-chain fatty acid (SCFA) concentrations to approximately the physiological levels noted after feeding. Plasma GH was markedly suppressed by feeding and at both the 107 and 214 mumol.kg-1.min-1 SCFA infusion rates; however, cholinergic blocking agents completely blocked the suppressed GH secretion after feeding and only at the 107 mumol.kg-1.min-1 infusion rate. Plasma glucose increased at both infusion rates, and the plasma free fatty acids decreased after feeding and at both infusion rates. However, both metabolites were unchanged relative to the saline control after the injection of the cholinergic antagonists. It is suggested that the decrease in plasma GH observed after feeding and a near-physiological ruminal SCFA increment is mediated via the parasympathetic nerve and not by pharmacological ruminal SCFA increments attributed to other pathways.

Mechanisms of action of growth hormone-releasing peptide-2 in bovine pituitary cells.

We conducted this study to investigate the mechanisms of action of growth hormone-releasing peptide-2 (D-Ala-D-beta Nal-Ala-Trp-D-Phe-Lys-NH2; GHRP-2) in bovine anterior pituitary primary cell culture. Doses of GHRP-2 from 10(-13) to 10(-7) M) increased (P < .05) GH secretion. The GHRP-2 (10(-7) M) and GH-releasing factor (GRF; 10(-7) M) administered together had an additive effect on the release of GH (P < .05). Somatostatin (1 microM) decreased GH secretion in response to GHRP-2 and(or) GRF (P < .05). Secretion of GH in response to GHRP-2 was blocked (P < .01) by a GRF receptor antagonist (.1 microM). Nifedipine (10 microM), a voltage-dependent Ca2+ channel blocker, inhibited (P < .01) GHRP-2-stimulated GH release. The GH release in response to GHRP-2 and 4 beta-phorbol-12-myristate-13-acetate (10(-7) M), a protein kinase C activator, was additive (P < .01). Forskolin (30 microM), a cAMP elevating agent, further stimulated (P < .01) the GH release in response to GHRP-2. Bovine GH concentrations in culture media were assayed by indirect competitive enzyme immunoassay. These results showed that GHRP-2 1) stimulates GH secretion from bovine pituitary cells, 2) may partially act via GRF receptor, 3) has GH secretion activity caused by Ca2+ influx via Ca2+ channels, and 4) may increase GH secretion via protein kinase C and cAMP pathways.

No desensitization of the growth hormone (GH) response between GH-releasing peptide-2 and GH-releasing factor in calves.

We investigated the desensitization caused by growth hormone (GH)-releasing peptide-2 (D-Ala-D-beta Nal-Ala-Trp-D-Phe-Lys-NH2; GHRP-2) in vivo using calves. The GHRP-2 (12.5 micrograms.kgBW-1.h-1) or GH-releasing factor (GRF; .125 microgram.kgBW-1.h-1) were infused for 180 min into four calves, each followed by either GHRP-2 (12.5 micrograms/kg BW) or GRF (.125 microgram/kg BW) injection after 60 min. All treatments were given to all of the calves. The continuous infusion of GHRP-2 did not attenuate the GH secretion caused by a GRF bolus, and vice versa. Continuous exposure to GHRP-2 attenuated the GH secretion caused by a GHRP-2 bolus. In an additional experiment, two repetitive injections of GHRP-2 (12.5 micrograms/kg BW) at 1-, 2-, 3-, or 4-h intervals were administered. Attenuation of the GH response caused by GHRP-2 was maintained until 4 h. These results suggest that GHRP-2 is not cross-desensitized with GRF and that the mechanisms by which they cause GH release may be different in calves.

Competitive enzyme immunoassay for bovine growth hormone.

We developed an enzyme immunoassay (EIA) for bovine GH (bGH) which is based on indirect competitive immunoassay in culture medium from a bovine pituitary cell culture. 40 microliters cell culture samples (or bGH standard) and bGH antibody (rabbit anti-bGH) were added to the 96 well microplate coated with secondary antibody (Goat anti-rabbit IgG), and incubated for 24 h at 37 degrees C. Biotin-label bGH was added and incubated further for 24 h at 37 degrees C, and biotinylated bGH was linked with streptoavidin-peroxidase. Substrates for peroxidase were added to the plate and incubated for 1 h at 4 degrees C. The enzyme reaction was stopped with 4N H2SO4, and the absorbency at 450 nm was measured with an ELISA Reader. The coefficients of intra-assay and inter-assay variations were 4.13 approximately 7.59% and 3.71 approximately 8.27%, respectively. The regression equation and correlation coefficients with the radioimmunoassay (RIA) were y(RIA) = 1.9986 x (EIA) - 1.3921 and 0.9701 (n = 27), respectively. Collectively, the present assay provides a reliable alternative to RIA and offers the major advantage of eliminating radioactive reagents and counting equipment.

Effects of muscarinic and adrenergic blockade on growth hormone secretion induced by growth hormone-releasing peptide-2 (GHRP-2) in ovariectomized ewes.

This study was performed to investigate whether the blockade of cholinergic muscarinic and adrenergic pathways was involved in the GH-releasing effect of GH-releasing peptide-2 (GHRP-2) in ovariectomized ewes. Cholinergic muscarinic antagonist, atropine (0.2 mg/kg, i.v., 15 min before GHRP-2 administration) blunted the GH secretion caused by GHRP-2 (12.5 microg/kgBW). alpha-Adrenergic antagonist phentolamine (15 microg/kgBW x min, infusion from -15 to 30 min) did not affect the GH response to GHRP-2, and beta-adrenergic antagonist propranolol (0.25 mg/kgBW, i.v., 15 min before GHRP-2 administration) did not suppress the GHRP-2-induced GH release. These results showed that cholinergic muscarinic antagonist agent, atropine, exerts an inhibitory effect on GHRP-2-induced GH secretion in ovariectomized ewes.

Effect of mesenteric venous volatile fatty acids (VFA) infusion on GH secretion in sheep.

The effects of mesenteric venous infusion of acetate, propionate and butyrate mixture (20.3, 40.5 and 81.0 micromol kg[-1] min[-1] over 4 h) on the secretion of GH was examined to investigate the effects of an increase in portal volatile fatty acids (VFA) on GH secretion in relation to inhibition of GH secretion after feeding in sheep. The mesenteric venous infusion at the rate of 40.5 micromol kg(-1) min(-1) increased the portal plasma VFA concentration within the approximate physiological range after feeding. Plasma GH was noticeably suppressed only at the infusion rate of 81.0 micromol kg(-1) min(-1) and the change in the mean concentration from the base line was significantly less than in the control. Although GRF injection rapidly increased plasma GH, the change in the mean concentration from the base line tended to suppress only at the infusion rate of 81.0 micromol kg(-1) min(-1). Plasma FFA was suppressed in a dose-dependent manner after VFA infusion. The change in the mean concentration from the base line was significantly suppressed only at the infusion rate of 81.0 micromol kg(-1) min(-1) relative to the control infusion, but plasma glucose was unchanged by VFA infusion. It is concluded that because the increase in the portal plasma VFA concentration within the range of feeding did not suppress GH secretion, VFA absorbed by the digestive tract may not play a significant role in suppressing GH secretion after feeding in sheep.

Effect of different experimental conditions on lipogenesis and substrate oxidation in isolated adipocytes from fattening Holstein steers.

The present study was undertaken to provide further information on lipogenesis in isolated adipocytes from the subcutaneous adipose tissue of fattening steers. The main aims were to compare different incubation media and to clarify the effects of fetal bovine serum (FBS) on lipogenesis and substrate oxidation rate in the adipocytes. The isolated adipocytes were prepared by the collagenase digestion technique. The changes in cellularity, the incorporation rates of acetate or glucose into lipid molecules, and the oxidation rates to CO2 both in fresh and preincubated adipocytes were measured in different media. It was shown that FBS increased significantly both lipogenesis and the oxidation rates of glucose or acetate in the fresh adipocytes but insulin did not. In isolated adipocytes preincubated for 24 and 48 h, the cellularity, the incorporation rate of glucose into lipid molecules, and the oxidation rates of glucose or acetate to CO2 were not different to those in fresh adipocytes, but the incorporation rate of acetate into lipid molecules was significantly lower.

Characteristics of growth hormone secretion responsiveness to growth hormone-releasing peptide-2 (GHRP-2 or KP102) in calves.

This study was conducted to determine the effects of acute and chronic administration of GH-releasing peptide-2 (D-Ala-D-beta Nal-Ala-Trp-D-Phe-Lys-NH2, GHRP-2 or KP102) on GH responsiveness in male Holstein calves. In the dose response study of acute administration, six calves were injected iv with saline or 6.25, 12.5 and 25.0 micrograms/kg body weight (BW) of KP102. The GH AUC (area under curve, ng/ml.min, mean +/- SEM) for 60 min was significantly increased with 6.25 (676.3 +/- 125.6), 12.5 (1574.8 +/- 318.0) and 25.0 (1578.7 +/- 214.6) micrograms/kgBW of KP102 than with saline (78.6 +/- 36.1) (P < 0.01). GH responses were decreased by multiple injections of 12.5 micrograms/kgBW KP102 at every 2 h for 8 h. The GH AUC for 60 min was decreased from the first injection (1162.9 +/- 313.3) to the second injection (604.7 +/- 131.9), but the response was significantly higher for the first and second injections than the third (304.4 +/- 173.1) and fourth injections (320.7 +/- 144.2) (P < 0.05). In the chronic administration, 8 calves were implanted subcutaneously with osmotic pumps (Alzet pump). Each of the 4 calves was given with 12.5 micrograms/kgBW per hour KP102 and the other 4 calves served as the control. During the 14 day period, average daily gain was significantly increased (36.4%) over the control (P < 0.05). Food efficiency was not significant, but numerically higher (29.4%) than the control. The plasma GH concentration was not increased by chronic administration of KP102, but IGF-I appeared to increase in KP102-treated calves more than the control. These results suggest that the synthetic KP102 can be used for enhancing the growth performance in domestic animals.