Preparation of an epithelium-free mammary fat pad and subsequent mammogenesis in ewes

The objective of this study was to develop a surgical procedure for the preparation of an epithelium-free mammary fat pad (cleared mammary fat pad; CFP) in ewes. At 7 to 10 d of age, ewe lambs (n = 43, mean BW 9.2 +/- .2 kg at 14 d) were sedated and one mammary gland was locally anesthetized. An incision circumscribing the base of the teat was made and blunt dissection was performed through the extraneous mammary fat pad tissue to enable the parenchyma and teat to be wholly removed. Failure to completely remove the epithelium enabled it to regenerate and grow into the mammary fat pad. Mean diameter of the parenchymal rudiment at 7 to 10 d of age was 8.9 +/- .5 mm (range of 5 to 16 mm). The excision site was closed with wound clips and recovered lambs returned to their dams. The contralateral mammary gland remained intact, allowing it to undergo normal development. Live weight gain was unaffected by this procedure. Ewes were subsequently slaughtered in groups at various stages of prepuberty, puberty, gestation, and lactation. Of 39 operated glands recovered, only one demonstrated epithelial outgrowth within the CFP. Parenchyma within the contralateral, intact gland underwent phases of rapid growth in prepuberty, puberty, and late gestation and was capable of milk synthesis after steroid induction or parturition. Change in weight of the CFP paralleled that of the intact mammary gland to 100 d of pregnancy. Sham CFP surgery was performed on four additional ewes wherein the parenchyma was completely excised and immediately replaced. Sham-operated epithelium populated the mammary fat pad and synthesized milk that could be expressed from the teat. A CFP in sheep will be a useful model for future investigations into the local growth regulatory mechanisms associated with ruminant mammogenesis.     

Growth hormone suppresses the expression of IGFBP-5, and promotes the IGF-I-induced phosphorylation of Akt in bovine mammary epithelial cells

Growth hormone (GH) plays a specific role to inhibit apoptosis in the bovine mammary gland through the insulin-like growth factor (IGF)-I system, however, the mechanism of GH action is poorly understood. In this study, we show that GH dramatically inhibits the expression of IGFBP-5, and GH along with IGF-I enhanced the phosphorylation of Akt through the reduction of IGF binding protein (IGFBP)-5. To determine how GH affects Akt through IGF-I in bovine mammary epithelial cells (BMECs), we examined the phosphorylation of Akt in GH treated BMECs and found that IGF-I induced phosphorylation of Akt was significantly enhanced by the treatment with GH. We demonstrated that GH reduces mRNA and protein expression of IGFBP-5 in BMECs, but it does not affect the expression of IGFBP-3. To determine that the enhanced effect of the Akt phosphorylation by the treatment of GH is due to the inhibition of the expression of IGFBP-5, we examined the effect of IGFBP-3 and -5 on the phosphorylation of Akt through IGF-I in the GH-treated BMECs. The phosphorylation of Akt was inhibited in a dose-dependent manner when IGFBP-5 was added at varying concentrations and was also inhibited in the presence of IGFBP-3. The results of this study suggest that GH plays an important role on mammary gland involution in bovine mammary epithelial cells.     

不同浓度胰岛素样生长因子-Ⅰ对体外培养奶牛乳腺上皮细胞乳蛋白和脂肪合成相关基因表达的影响

应用Real-time PCR方法检测不同浓度(0、1、10和100 ng/mL)胰岛素样生长因子-Ⅰ(IGF-Ⅰ)处理后的体外培养奶牛乳腺上皮细胞乳蛋白和脂肪合成相关基因mRNA的相对表达量。结果表明,添加不同浓度IGF-Ⅰ对体外培养的奶牛乳腺上皮细胞IGF-Ⅰ受体基因(IGFIR)、IGF-Ⅰ结合蛋白-3基因(IGFBP3)、α-s1-酪蛋白基因(CSN1S1)和κ-酪蛋白基因(CSN3)mRNA的相对表达丰度均无显著影响(P>0.05)。随着IGF-Ⅰ添加浓度的增加,β-酪蛋白基因(CSN2)、乙酰辅酶A羧化酶基因(ACACA)、脂肪酸合成酶基因(FASN)和脂肪酸结合蛋白-3基因(FABP3)mRNA的相对表达丰度显著上调(P<0.05)。提示,IGF-Ⅰ作为一种重要细胞因子参与调节乳腺上皮细胞乳蛋白和乳脂肪相关基因mRNA的表达。     

不同泌乳相关激素和生长因子对奶牛乳腺上皮细胞增殖的影响

本研究旨在探讨不同泌乳相关激素和生长因子对奶牛乳腺上皮细胞增殖的影响及其与细胞外基质主要成分层黏连蛋白的关系。将正常的荷斯坦泌乳期奶牛乳腺上皮细胞进行体外培养,在未包被或包被层黏连蛋白的条件下,以MTT法检测催乳素(PRL)、牛生长激素(GH)、类胰岛素生长因子-1(IGF-Ⅰ)、类胰岛素生长因子-2(IGF-Ⅱ)对细胞增殖作用的影响。在层黏连蛋白包被条件下,进行血清恢复的同时添加不同泌乳相关激素和生长因子,GH、IGF-Ⅰ有促进细胞增殖的作用(P<0.05),PRL、IGF-Ⅱ有维持细胞存活的作用(P<0.05);无血清时,几种激素和生长因子单独添加均无明显促增殖效应(P>0.05)。无基质条件下,与血清联合使用时,PRL、GH、IGF-Ⅰ、IGF-Ⅱ均对细胞生长有不同程度促进作用(P<0.05);无血清时,仅IGF-Ⅰ使细胞增殖速率显著加快(P<0.05)。层黏连蛋白作为培养基质对于体外培养的泌乳乳腺上皮细胞生长速度没有显著促进作用,但有利于PRL和IGF-Ⅱ发挥促存活作用。PRL、GH、IGF-Ⅰ、IGF-Ⅱ对细胞增殖和存活有促进作用,但需要与血清中其他成分协同才能充分发挥作用,其中IGF-Ⅰ促增殖能力最强。     

Identification of IGF binding proteins in bovine milk and the demonstration of IGFBP-3 synthesis and release by bovine mammary epithelial cells.

Insulin-like growth factor system components are synthesized and secreted by mammary epithelial cells and multiple IGF binding proteins (IGFBP) are found in milk of various species. This study was conducted to identify the IGFBP in bovine milk, to compare them with those found in blood, and to identify the cell(s) responsible for mammary IGFBP synthesis. Bovine blood, milk, and cell culture-conditioned media were analyzed and characterized with Western ligand blot procedures for specific IGFBP. Electrophoresis and [125I]IGF-II ligand blot analyses of the samples indicated that, unlike serum and mammary primary cell culture-conditioned media, milk required removal of casein in order to accurately disclose all IGFBP. Immunoprecipitation studies identified IGFBP-2, -3, -4, and -5 in blood, milk, and primary cell culture conditioned media. The IGFBP were present at higher concentrations in serum than in milk, and milk concentrations were greater than that shown in conditioned media from primary cultures of bovine mammary cells. Northern analysis detected IGFBP-3 messenger RNA in extracts from fresh tissue and cells in culture, and in situ hybridization studies with fresh tissue utilizing probes for IGFBP-3 and alphaS1-casein showed that the mRNA for IGFBP-3 is predominant in the secretory epithelial cells, when compared to other tissue cell types.     

Effects of breed and wintering diet on growth, puberty and plasma concentrations of growth hormone and insulin-like growth factor 1 in heifers

Twenty-five Brangus (BR) and 15 Angus (AN) heifers were used to study the effects of breed and wintering diet on average daily gain (ADG), onset of puberty and plasma concentrations of growth hormone (GH) and insulin-like growth factor 1 (IGF-1). Wintering diets (fed for 107 days beginning November 15) consisted of the following: 1) native grass hay (NGH), 2) ammoniated NGH, 3) NGH plus cottonseed meal, 4) Diet 3 plus corn and 5) Diet 4 plus monensin. After wintering, heifers were transferred to ryegrass pasture for 70 days. Mean ADG during the wintering phase were -.20, -.10, .17, .29 and .39 kg for heifers fed Diets 1 through 5, respectively (P less than .01). ADG was greater (P less than .05) for BR than for AN heifers. Plasma concentrations of GH were higher (P less than .05) in heifers fed Diets 1 and 2 than in heifers fed Diets 3, 4 or 5. Plasma concentrations of IGF-1 were lowest in heifers fed Diet 1 and highest in heifers fed Diets 4 and 5. During ryegrass grazing, GH concentrations were similar for all groups. However, concentrations of IGF-1 were higher (P less than .05) in heifers fed Diets 3, 4 and 5 than in heifers fed Diets 1 and 2. Age at puberty (onset of cyclic progesterone concentrations) was greatest in heifers fed Diet 1 and lowest in heifers fed Diet 5. Weight at puberty was not affected (P greater than .10) by wintering diet but was greater (P less than .01) in BR than in AN heifers. Therefore, negative ADG appears to be associated with elevated plasma GH concentrations in heifers, and plasma IGF-1 concentration appears to be a more accurate indication of nutritional status than plasma concentrations of GH.     

Effects of growth factors on insulin-like growth factor binding protein (IGFBP) secretion by primary porcine satellite cell cultures.

Insulin-like growth factor-binding proteins (IGFBP) regulate the biological functions of insulin-like growth factors (IGF) and may affect cell growth through IGF-independent actions. Growth factors and hormones have been shown to alter IGFBP production by target cells suggesting that the effects of these factors may be partially mediated by the local production of IGFBP. Growth factors, including IGF-I, transforming growth factor-beta1 (TGF-beta1), and basic fibroblast growth factor (bFGF) have potent effects on satellite cell proliferation and differentiation, and some of these factors have been shown to alter IGFBP production in various cell types. Consequently, some of their actions on muscle satellite cells may be mediated by the local production of IGFBP. In this study, we measured the effects of IGF-I, bFGF, and TGF-beta1 on IGFBP production by primary porcine satellite cell (PSC) cultures after first determining physiologically active concentrations of these growth factors to use according to [3H]thymidine incorporation dose responses. There is little information on the effects of these growth factors on IGFBP production in primary porcine myogenic cells due to the confounding affects of contaminating nonmuscle fibroblasts. Comparative studies show that primary porcine satellite cells produce IGFBP-3 and -5 whereas porcine muscle-derived nonfusing cells (FIB) produce IGFBP-2 and -4 but not IGFBP-3 or -5. Because of this, our investigations have focused on growth factor-induced production of IGFBP-3 and -5 in primary porcine satellite cells cultures. Both IGF-I and bFGF exhibited dose-dependent increases in [3H]thymidine incorporation with increasing concentration from 1 to 50 ng/mL (P < 0.05), whereas TGF-beta1 caused a dose-dependent decrease from 0.01 to 0.5 ng/mL (P < 0.05). When 20 ng/ mL of IGF-I was added to the media, IGFBP-3 was increased approximately 65% (P < 0.05) and IGFBP-5 was increased approximately twofold (P < 0.05). The addition of 0.5 ng/mL TGF-beta1 caused more than a two-fold increase in IGFBP-3 (P < 0.05) and approximately an 80% increase in IGFBP-5 (P < 0.05), whereas 50 ng/ mL of bFGF caused approximately 40% (P < 0.05) and 70% (P < 0.05) increases in IGFBP-3 and -5, respectively. Neither IGFBP-3 nor -5 was detectable in the conditioned media from fibroblasts whether or not IGF-I, TGF- beta1 or bFGF were present. These data suggest that the effects of IGF-I, TGF- beta1 and bFGF on porcine satellite cells may in part be through the autocrine/ paracrine production of IGFBP-3 and -5 by porcine satellite cells.     

Evaluation of serum insulin-like growth factor binding proteins (IGFBP) in Angus cattle divergently selected for serum IGF-I concentration

Postweaning serum insulin-like growth factor-I (IGF-I) concentrations and serum IGF binding proteins (IGFBP) were investigated in 68 (1992 Fall-born) and 84 (1999 Fall-born) Angus cattle selected for either high or low serum IGF-I concentrations since 1989. Relative serum levels of IGFBP were determined by [¹²⁵I]IGF-I Western ligand blotting. IGFBP species of 38–42, 34, 30, and 24 kDa were identified. The 34 kDa species was identified as IGFBP-2 by immunoblot analysis. No significant line effects were observed for any of the IGFBP. In both 1992 and 1999, heifers had higher IGFBP-2 levels than bulls (P<0.0005). In 1992 calves, relative levels of the 38–42 and 24 kDa species were significantly correlated with serum IGF-I concentration. In 1999 calves, none of the IGFBP were correlated with serum IGF-I, although IGFBP-2 was negatively correlated with several measures of body weight. No significant line effects were observed for growth or serum IGF-I traits in 1992 calves. However, 1999 high line calves had higher serum IGF-I concentrations and body weights than low line calves (P<0.05). In both 1992 and 1999 calves, bulls had higher serum IGF-I concentrations and body weights than heifers (P<0.05). Thus, while selection for high versus low serum IGF-I concentrations has resulted in divergence between the selection lines and also in changes in body weights, it has not resulted in changes in serum IGFBP levels. Furthermore, circulating IGFBP-2 appears to be higher in heifers than in bulls, and also appears to be negatively correlated with body weights.     

Insulin-like growth factor binding proteins initiate cell death and extracellular matrix remodeling in the mammary gland

We have demonstrated that insulin-like growth factor binding protein-5 (IGFBP-5) production by mammary epithelial cells increases dramatically during forced involution of the mammary gland in rats, mice and pigs. We proposed that growth hormone (GH) increases the survival factor IGF-I, whilst prolactin (PRL) enhances the effects of GH by decreasing the concentration of IGFBP-5, which would otherwise inhibit the actions of IGFs. To demonstrate a causal relationship between IGFBP-5 and cell death, we created transgenic mice expressing IGFBP-5, specifically, in the mammary gland. DNA content in the mammary glands of transgenic mice was decreased as early as day 10 of pregnancy. Mammary cell number and milk synthesis were both decreased by approximately 50% during the first 10 days of lactation. The concentrations of the pro-apoptotic molecule caspase-3 was increased in transgenic animals whilst the concentrations of two pro-survival molecules Bcl-2 and Bcl-x were both decreased. In order to examine whether IGFBP-5 acts by inhibiting the survival effect of IGF-I, we examined IGF receptor- and Akt-phoshorylation and showed that both were inhibited. These studies also indicated that the effects of IGFBP-5 could be mediated in part by IGF-independent effects involving potential interactions with components of the extracellular matrix involved in tissue remodeling, such as components of the plasminogen system, and the matrix metallo-proteinases (MMPs). Mammary development was normalised in transgenic mice by R3-IGF-I, an analogue of IGF-I which binds weakly to IGFBPs, although milk production was only partially restored. In contrast, treatment with prolactin was able to inhibit early involutionary processes in normal mice but was unable to prevent this in mice over-expressing IGFBP-5, although it was able to inhibit activation of MMPs. Thus, IGFBP-5 can simultaneously inhibit IGF action and activate the plasminogen system thereby coordinating cell death and tissue remodeling processes. The ability to separate these properties, using mutant IGFBPs, is currently under investigation.     

Action of long(R)-insulin-like growth factor-1 on protein metabolism in beef heifers

Insulin-like growth factor-1 (IGF-1) is perhaps the most important endogenous factor controlling growth. Most studies to date in livestock have shown that IGF-1 has greatest efficacy when animals are in a catabolic state. We have determined the effects of an iv infusion of the IGF-1 analog Long(R³)-IGF-1 on protein metabolism in beef heifers that were slowly losing liveweight because of restricted feeding. There was a tendency for both whole-body protein and skeletal muscle protein to be conserved in Long(R³)-IGF-1-treated heifers. Long(R³)-IGF-1 administration markedly reduced the plasma concentrations of all amino acids measured and glucose. There was a significant change in the profile differences of endogenous plasma IGF-1 concentrations during the 8-hr infusion period, with plasma IGF-1 decreasing sharply in the test group. There was a significant difference in mean profiles for plasma IGF-2 between the test and control groups. Overall, plasma IGF-2 for the control group decreased only slightly over time (about 40 ng/ml), whereas the test group decreased dramatically (by about 140 ng/ml). Increased plasma concentrations of a 31–32-kDa IGF-binding protein (possibly IGF-binding protein-1) in the treated group was detected by radioligand blot. We found that Long(R³)-IGF-1 infusion tended to preserve whole-body and muscle protein in beef heifers on a low-quality diet, and suggest that further investigation of this treatment may provide an alternative approach to reducing weight loss during the dry season.     

Ovarian and endocrine characteristics during an estrous cycle in Angus, Brahman, and Senepol cows in a subtropical environment

To determine breed differences in ovarian function and endocrine secretion, daily rectal ultrasonography was conducted on multiparous lactating Angus (temperate Bos taurus; n = 12), Brahman (tropical Bos indicus; n = 12), and Senepol (tropical Bos taurus; n = 12) cows during an estrous cycle in summer. Blood was collected daily to quantify plasma concentrations of FSH, LH, progesterone, estradiol, GH, insulin-like growth factor (IGF)-I, IGF-II, IGF binding proteins (IGFBP), insulin, glucose, and plasma urea nitrogen (PUN). Numbers of small (2 to 5 mm), medium (6 to 8 mm), and large follicles (> or = 9 mm) were greater (P < .05) in Brahman than in Angus and(or) Senepol cows. Length of the estrous cycle (SEM = .6 d) was similar (P > .10) among Senepol (20.4 d), Angus (19.5 d), and Brahman (19.7 d) cows. Senepol cows had greater (P < .05) diameters of the corpus luteum (CL) and a delayed regression of the CL as compared with Angus cows. The secondary surge of FSH (between d 1 and 2; d 0 = estrus) was greater in Angus than Brahman or Senepol cows (breed x day, P < .05). Between d 2 and 14 of the estrous cycle, concentrations of progesterone, LH, IGF-II, and binding activities of IGFBP-3, IGFBP-2, and the 27- to 29-kDa IGFBP in plasma did not differ (P > .10) among breeds. Concentrations of GH, IGF-I, insulin, and PUN were greater (P < .001) and binding activities of the 22-kDa and 20-kDa IGFBP tended (P < .10) to be greater in plasma of Brahman than in Angus or Senepol cows. Plasma glucose concentrations were greater (P < .05) in Senepol than in Brahman or Angus cows. In conclusion, Brahman (Bos indicus) and Senepol cows (tropical Bos taurus) had greater numbers of follicles in all size categories and greater diameter of CL than Angus (temperate Bos taurus) cows. These ovarian differences may be due to changes in the pattern of secretion of FSH, insulin, IGF-I, and GH but not LH, IGF-II, or IGFBP-2 or -3.     

IGF-1 stimulates protein synthesis by enhanced signaling through mTORC1 in bovine mammary epithelial cells

Using the MAC-T cell line as a model, the effects of insulin-like growth factor (IGF)-1 on the regulation of protein synthesis through the mammalian target of rapamycin complex 1 (mTORC1) signaling in bovine mammary epithelial cells were evaluated. Global rates of protein synthesis increased by 47% within 30 min of IGF-1 treatment. The effect of IGF-1 on protein synthesis was associated with enhanced association of the eukaryotic initiation factor (eIF) 4E with eIF4G and a concomitant reduction of eIF4E association with eIF4E-binding protein-1 (4E-BP1). There was a progressive increase in the phosphorylation state of ribosomal protein S6 kinase-1, a downstream target of mTORC1 in response to IGF-1. In addition, IGF-1 stimulated mTORC1 kinase activity toward 4E-BP1 in vitro. Phosphorylation on Ser473 of Akt was induced by IGF-1 within 5 min and remained elevated throughout a 30-min time course. The effect of IGF-1 on Akt phosphorylation was also concentration dependent. Activation of Akt by IGF-1 led to increased phosphorylation of tuberous sclerosis complex 2 on Thr1426, without any change in its association with tuberous sclerosis complex 1. Phosphorylation of proline-rich Akt substrate of 40-kDa (PRAS40) at Thr246 was stimulated by IGF-1. The amount of PRAS40 associated with mTORC1 decreased in response to IGF-1, and PRAS40 binding to mTORC1 was inversely related to its phosphorylation level. Overall, these results suggest that activation of the PI3K-Akt pathway by IGF-1 stimulated global protein synthesis in bovine mammary epithelial cells through changes in the phosphorylation and association state of components of the mTORC1 signaling pathway.     

Plasma hormone and metabolite concentrations involved in the somatotropic axis of Japanese Black heifers in association with growth hormone gene polymorphism

Bovine growth hormone (bGH) gene polymorphism of leucine (Leu)-threonine (Thr) (allele A), valine (Val)-Thr (allele B), and Val-methionine (Met) (allele C) at codons 127 and 172 was shown to relate with carcass trait variations in Japanese Black cattle. In this study, 10-mo-old Japanese Black heifers with growth hormone (GH) genotypes AA, AB, BB, AC, BC, and CC (N = 141) were compared for basal GH, insulin-like growth factor-1 (IGF-1), insulin, ghrelin, glucose, and nonesterified fatty acid (NEFA) concentrations. Growth hormone release was also measured as response to growth hormone–releasing hormone (GHRH) (0.4 μg/kg body weight [BW]) using 18 heifers with GH genotypes AA, BB, and CC (n = 6 for each group). The genotype AA heifers showed the greatest BW among genotypes (P < 0.05). Genotype AC, BC, and CC heifers showed greater GH concentrations than genotype AA, AB, or BB heifers, in which genotype CC heifers had the highest concentrations (P < 0.05). However, IGF-1 concentrations did not significantly differ. The genotype AA and BB heifers had a greater GH release at 60 min following GHRH injection than did the genotype CC heifers. The area under the curve (AUC; P < 0.07) and incremental area (IA; P < 0.08) of GH responses to the GHRH challenge tended to be the highest in the genotype AA heifers and the lowest in the genotype CC heifers. In conclusion, GH gene polymorphism altered GH, which may have contributed to differences in BW and carcass traits among genotypes.     

Effect of feed restriction on serum somatotropin, insulin-like growth factor-I-(IGF-I) and IGF binding proteins in cyclic heifers actively immunized against growth hormone releasing factor

Feed restriction often increases serum somatotropin (ST) and decreases insulin-like growth factor-I (IGF-I) in ruminants; however, the mechanisms responsible for this change in ST and IGF-I are not well defined. We investigated the effects of feed restriction on serum ST, IGF-I, IGF binding proteins (IGFBP), insulin and nonesterified fatty acids (NEFA) in cyclic Angus and Charolais heifers (n=15) previously immunized against growth hormone releasing factor (GRFi) or human serum albumin (HSAi). Cows were fed a concentrate diet ad libitum (AL) or were restricted to 2 kg cotton seed hulls (R) for 4 d. Each heifer received each dietary treatment in a single reversal design. As anticipated, GRFi decreased ST, IGF-I and insulin (P<.05). In addition, GRFi decreased serum IGFBP-3 (P<.01), but increased IGFBP-2 (P<.01). Feed restriction resulted in an increase in serum ST in HSAi, but not in GRFi heifers. Regardless of immunization treatment, feed restriction decreased serum IGF-I and insulin, and increased NEFA (P<.01). In conclusion, the increase in serum ST levels observed during feed restriction was blocked by active immunization against GRF. However, feed restriction resulted in decreased serum IGF-I in GRFi heifers in spite of initial low levels of IGF-I (due to GRFi). Although GRFi decreased levels of IGFBP-3 and increased levels of IGFBP-2, feed restriction for 4 d did not alter serum IGFBP.     

Possible role of growth hormone, IGFs, and IGF-binding proteins in the regulation of ovarian function in large farm animals.

The aim of the study and short review was to present evidence that growth hormone (GH), locally produced insulin-like growth factors (IGFs), and IGF-binding proteins (IGFBPs) may have an important role in the control of ovarian function. There is clear evidence for a distinct GH-receptor mRNA expression and protein production in follicles (oocytes and granulosa-cumulus cells) and corpus luteum (CL). In hypophysectomized ewes, GH and LH are necessary for normal CL development. IGF-1 mRNA in the follicles is expressed in theca interstitial cells (TIC) and granulosa cells (GC) with already higher levels in the TIC before follicle selection. In contrast, IGF-2 is mainly expressed in the TIC. The IGFR-1 mRNA is expressed in both the TIC and GC, with increasing levels in GC during the final development of dominant follicles. IGF-1 is a very potent stimulator of progesterone and oxytocin release in GC. IGFBP-1, -2, -3, -4, -5, and -6 have been isolated from follicular fluid or ovarian tissue. Studies indicate that IGFBP expression and production in the developing follicle is dependent on both cell type and follicle size and is regulated by IGF-1 and gonadotropins. The highest expression of IGF-1 and IGFR-1 mRNA was demonstrated during the early luteal phase. Distinct receptors for IGF-1 and IGF-2 were present in CL membrane preparations at all stages investigated. Intense immunostaining for IGF-1 was observed mainly in bovine large and small luteal cells and in a limited number of endothelial cells. In contrast, IGF-2 protein was localized in perivascular fibroblast and pericytes of the capillaries. With the use of a microdialysis system, we found that in vitro and in vivo IGF-1, IGF-2, and GH stimulated the release of progesterone in cultures of luteal cells or intact tissues. In conclusion, there is clear evidence for a central role of the IGFs, IGFBPs, and GH in follicular development and CL function.     

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 weaning on somatotrophic gene expression and circulating levels of insulin-like growth factor-1 (IGF-1) and IGF-2 in pigs

The present study evaluated somatotrophic gene expression in liver, muscle and adipose tissue 4 d after weaning, a time point corresponding to greatly reduced serum concentrations of insulin-like growth factor (IGF)-1 and IGF-2 in pigs. Two-week-old barrows were either cross-fostered to a sow (SOW, N = 8) or weaned and fed a phase 1 diet containing either 0 or 7% spray-dried plasma (NP, N = 8 and SDP, N = 8; respectively). Piglets were allocated such that two size groups were equivalently represented in each experimental group (small, 3.5–4.3 kg and large, 4.6–5.7 kg). Animals were weighed daily and sacrificed 4 d after weaning for blood and tissue collection. Daily gains of the SOW piglets were significantly greater than those of the weaned pigs for the first 3 d of the experiment (P < 0.0001). Weight gains in the SOW and SDP pigs between d 3 and 4 were equivalently elevated relative to the NP pigs (P < 0.0001). Serum IGF-1 and IGF-2 concentrations were decreased in both NP and SDP compared to SOW (P < 0.0001). Serum IGF-2 levels were significantly lower in small piglets (P = 0.006). A Weaning Group X Size interaction was noted for liver IGF-2 mRNA (P < 0.03), reflecting a higher level of expression in large SOW piglets relative to small SOW piglets. Weaning did not affect IGF-1, IGF-2, or growth hormone (GH) receptor mRNA levels in liver, muscle, or fat (P > 0.05). Liver IGF-binding protein (IGFBP)-3 and acid-labile subunit (ALS) mRNA levels also were unaffected by weaning. Small pigs had lower levels of liver ALS (P = 0.0003), muscle IGF-2 (P = 0.02), and muscle GH receptor (P = 0.006) mRNAs. In contrast, adipose tissue IGF-1 and IGF-2 mRNA levels were greatest in the small piglets (P = 0.001 and 0.029, respectively).     

Predicting growth in angus bulls: the use of GHRH challenge, insulin-like growth factor-I, and insulin-like growth factor binding proteins

Plasma IGF-I, IGF binding protein-2 (IGFBP-2), and IGFBP-3 were quantified in growing Angus bulls (n = 56) to determine their relationship with postweaning growth and carcass ultrasound measurements. In addition, GH response to GHRH challenge (area-under-the-curve GH [AUC-GH) was determined for each bull as part of a previous study. Blood was collected by jugular venipuncture at the start of a 140-d postweaning growth performance test and at 28 d intervals for plasma IGF-I determination by RIA. Plasma IGFBP-2 and -3 content was measured at the start of the study, on d 70, and d 140 by Western ligand blotting. Individual weights and hip heights were measured every 28 d during the study and carcass longissimus muscle area, intramuscular fat percentage, and carcass backfat were estimated by ultrasound on d 140. Greater plasma IGF-I at the start of the performance test was associated with reduced postweaning ADG and increased longissimus area. Throughout the performance test period, the correlations between plasma IGF-I and hip height were consistently positive, ranging from 0.10 to 0.38, but the correlations between ADG and IGF-I varied from -0.32 to 0.31. Age-adjusted d-1 plasma IGFBP-2 was related to ADG during the performance test, explaining nearly 30% of the variation in ADG. A model combining weaning age, IGFBP-2, and AUC-GH showed a strong relationship with ADG (R2 = 0.40). Plasma IGFBP-2 and -3 were not related to carcass characteristics, and IGFBP-3 was not related to growth rates. This study provides additional evidence for the variable relationship between plasma IGF-I and growth rates in cattle. A significant positive relationship between plasma IGFBP-2, AUC-GH, and postweaning ADG warrants further investigation.     

Effect of increasing levels of undegradable intake protein on metabolic and endocrine factors in estrous cycling beef heifers

To determine the influence of three levels of undegradable intake protein (UIP) supplementation on metabolic and endocrine factors that influence reproduction, 23 yearling crossbred heifers (body condition score = 4.5 +/- 0.5; initial BW = 362 +/- 12 kg) were stratified by BW and assigned randomly to one of three supplements: 1) low UIP (1,135 g x heifer(-1) x d(-1); 30% CP, 115 g UIP, n = 7); 2) mid UIP (1,135 g x heifer(-1) x d(-1); 38% CP, 216 g UIP, n = 8); or 3) high UIP (1,135 g x heifer(-1) x d(-1); 46% CP, 321 g UIP, n = 8). Heifers were estrually synchronized before initiation of supplementation. Supplement was individually fed daily for 30 to 32 d, at which time heifers were slaughtered (d 12 to 14 of the estrous cycle) and tissues collected. Heifers were fed a basal diet of sudan grass hay (6.0% CP) ad libitum. On d 28 of supplementation (d 10 of the estrous cycle), no differences were observed (P > 0.10) in serum insulin or IGF-I among treatments. At slaughter (d 10 to 12 of the estrous cycle), treatments did not influence corpus luteum weight, cerebral spinal fluid leptin, or IGFBP; serum estradiol-17beta, progesterone, leptin, IGF-I, and IGFBP; or anterior pituitary content of IGFBP (P > 0.10). Follicular fluid IGFBP-2 and IGFBP-4 were greater in high-UIP heifers than low- or mid-UIP heifers on d 12 to 14 of the estrous cycle (P < 0.05). Basal serum LH concentrations and LH area under the curve (every 15 min for 240 min) did not differ (P > 0.10) following 28 d of supplementation (d 10 of the estrous cycle); however, basal serum FSH concentrations were greater (P = 0.06) in low- and mid- vs. high-UIP heifers (5.2 and 5.2 vs. 4.6 ng/mL, respectively), and FSH area under the curve was greater (P = 0.03) in low- vs. high-UIP heifers. At slaughter (d 12 to 14 of the estrous cycle), anterior pituitary LH and FSH content and steady-state mRNA encoding alpha, LHbeta, and GnRH receptor did not differ (P > 0.10) among treatments. However, FSHbeta mRNA was increased approximately twofold (P = 0.03) in mid vs. low UIP. In summary, low and mid levels of UIP supplements fed to estrous cycling beef heifers seemed to enhance pituitary expression and/or secretion of FSH relative to high levels of UIP. Moreover, high-UIP supplementation was associated with increased low-molecular-weight IGFBP compared with supplementation of low and mid levels of UIP. These data suggest that differing levels of UIP supplementation may alter pituitary and ovarian function, thereby influencing reproductive performance in beef heifers.