Age-related changes in serum insulin-like growth factor-I, insulin-like growth factor-I binding protein-3 and articular cartilage structure in Thoroughbred horses

REASONS FOR PERFORMING STUDY: Structural changes in articular cartilage associated with the ageing process require definition for investigators performing developmental and age-related studies, for which information is lacking. OBJECTIVES: To 1) determine the onset and end of puberty as defined by serum insulin like growth factor (IGF-I) and IGF-binding protein-3 (IGFBP-3) concentrations and 2) correlate articular-epiphyseal cartilage complex structural changes with the onset and end of puberty. METHODS: IGF-I and IGFBP-3 were measured in serum samples from normal female and male horses age 9-715 days to determine peak and steady-state values for horses transitioning through puberty. Osteochondral tissue sections were obtained from horses age 120-840 days (4-28 months) and examined histologically for cartilage canals and tidemark formation. RESULTS: In male and female horses, serum IGF-I/IGFBP-3 concentrations peaked at approximately 225 days, defining the onset of puberty. Cartilage canals were absent from articular cartilage just prior to this time point. IGF-I/IGFBP-3 concentrations declined to steady-state levels at approximately age 450 days, signalling exit from puberty and therefore the beginning of ageing. This time point correlated to initial formation of a tidemark in the osteochondral tissue sections. CONCLUSIONS: Horses may be considered pubescent at age 225-450 days, and post pubescent and ageing after age 450 days. POTENTIAL RELEVANCE: Defining the normal post natal to post pubescent concentrations for serum IGF-I and serum IGFBP-3 establishes subsets of animals for age-related studies and may be used to monitor horses for abnormally high IGF-I concentrations due to natural disease or subsequent to systemic growth hormone administration.     

Immunolocalization of insulin-like growth factor-I (IGF-I) and its receptors (IGF-IR) in the equine epididymis

Insulin-like growth factor plays a paracrine/autocrine role in regulating testicular function in the stallion, but its presence in the equine epididymis remains unknown. The aim of this study was to test the hypothesis that insulin-like growth factor-I (IGF-I) and IGF-I receptor (IGF-IR) are localized in the caput, corpus, and cauda of the epididymis in an age-dependent manner. Immediately after castration, epididymal tissue was fixed, paraffin-embedded, and processed for immunohistochemistry (IHC). Western blot was also performed using equine epididymal extracts to verify the specificity of the antibodies against IGF-I and IGF-IR. Immunolabeling of IGF-I was observed in the cytoplasm of principal and basal cells in the caput, corpus, and cauda at the pre-pubertal (3–7 months), pubertal (12–18 months), post-pubertal (2–4 years), and adult stages (4.5–8 years). Immunolabeling of IGF-IR was observed in the cytoplasm of principal cells in all regions of the epididymis in each age group. Immunolabeling of IGF-IR was also detected in the cytoplasm of basal cells from animals of all ages. Bands observed by Western blot corresponded to the molecular weights of IGF-I and IGF-IR, ~23 kDa and 95 kDa, respectively. These results suggest that IGF-I might function as an autocrine and/or paracrine factor during the development, maintenance and/or secretions of the stallion epididymis.     

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.     

Epitope-tagged insulin-like growth factor-I expression in muscle

Development of a recombinant insulin like growth factor I (IGF-I) that is distinguishable from its endogenous counterpart would provide a powerful tool for delineating the role of IGF in myogenesis. Therefore, the objective of this study was to create an epitope-tagged IGF-I that retains biological activity and determine whether expression of this construct is possible in muscle tissue following direct DNA injection. Expression vectors were created that encoded porcine IGF-I containing a T7 (11-amino acid) epitope-tag (TIGF). Immunoreactivity of the purified recombinant TIGF was confirmed using monoclonal antibodies. Biological activity was evaluated by examining differentiation of myoblasts cultured with TIGF or transfected with TIGF plasmid DNA. Addition of purified TIGF to myoblast cultures stimulated (P < 0.05) muscle creatine kinase levels similar to insulin (10(-5) M). Likewise, transfection of L6A1 with TIGF DNA hastened (P < 0.01) differentiation compared to control pcDNA-transfected myoblasts. The integrity of the recombinant protein was confirmed using a sandwich-configured enzyme linked immunosorbent assay. Finally, recombinant TIGF DNA was injected in porcine muscle and the ability to detect TIGF protein was evaluated. TIGF expression was detected in muscle fibers of injected porcine muscle. These data show that a T7 amino acid tag placed on the amino terminus of the IGF-I protein remains intact during processing and does not interfere with the biological activity of the molecule. Use of this DNA construct is an excellent tool for investigating the role of IGFs in control muscle development and provides a model to investigate other regulators of animal growth.     

Effects of beta-aminopropionitrile on equine tendon metabolism in vitro and on effects of insulin-like growth factor-I on matrix production by equine tenocytes

OBJECTIVE: To investigate effects of beta-aminopropionitrile and a combination of insulin-like growth factor (IGF)-I and beta-aminopropionitrile on metabolism of equine tendon fibroblasts. SAMPLE POPULATION: Flexor tendon explants from 3 horses. PROCEDURE: Explants received 1 of 4 treatments (control, IGF-I, beta-aminopropionitrile, and IGF-I/beta-aminopropionitrile) for 10 days, and message expression for collagen types I and III was assessed by use of in situ hybridization. Histologic findings, new protein production, and quantitative determinations of glycosaminoglycan, DNA, and de novo collagen synthesis were made. RESULTS: Insulin-like growth factor-I stimulated an anabolic response in tendon. Collagen synthesis and glycosaminoglycan and DNA content of explants were all increased. Beta-aminopropionitrile significantly suppressed collagen synthesis, which was not ameliorated by concurrent IGF-I treatment. Beta-aminopropionitrile caused alterations in cell morphology characterized by large round cells with eccentric nuclei and decreased density of collagen fibers. Protein production and collagen type-III mRNA expression were reduced in these cells. CONCLUSIONS AND CLINICAL RELEVANCE: Treatment with beta-aminopropionitrile resulted in decreased production of protein and collagen synthesis, which could be expected to suppress tendon healing. The negative effects of beta-aminopropionitrile could not be abrogated by addition of IGF-I to the medium. Treatment resulted in alterations in cell morphology and matrix consistency, which could further delay tendon healing. Beta-aminopropionitrile may impair tendon healing at a cellular level by decreasing collagen production or increasing rate of degradation of existing matrix. Because of reduced crosslinking during beta-aminopropionitrile treatment, in combination with transiently decreased tensile strength, alterations in collagen content and structure may weaken the healing tendon.     

Expression of the ACTH receptor, steroidogenic acute regulatory protein, and steroidogenic enzymes in canine cortisol-secreting adrenocortical tumors

Studies of human adrenocortical tumors (ATs) causing Cushing's syndrome suggest that hypersecretion of cortisol is caused by altered expression of steroidogenic enzymes and that steroidogenesis can only be maintained when there is expression of the ACTH receptor (ACTH-R). Here we report the screening for the mRNA expression of the ACTH-R, steroidogenic acute regulatory protein (StAR), cholesterol side-chain cleavage enzyme, 3β-hydroxysteroid dehydrogenase, 21-hydroxylase (all in 38 cortisol-secreting ATs), 17α-hydroxylase, and 11β-hydroxylase (both in 28 cortisol-secreting ATs). Real-time PCR (RT-PCR) was applied in all samples and was compared with that in normal canine adrenal glands. Messenger-RNA encoding StAR, steroidogenic enzymes, and ACTH-R were present in both normal adrenal glands and cortisol-secreting ATs. The amounts of mRNA encoding StAR and enzymes of the steroidogenic cluster needed for cortisol production did not differ significantly between either adenomas or carcinomas and normal adrenal glands. The amount of mRNA encoding ACTH-R was significantly lower in carcinomas than in normal adrenal glands (P = 0.008). In conclusion, RT-PCR analysis revealed no overexpression of StAR and steroidogenic enzymes in canine cortisol-secreting ATs. Significant downregulation of ACTH-R in carcinomas might be associated with the malignant character of the AT.     

Peripheral circulating insulin-like growth factor-I and -II in cattle

Interrelationships between circulating concentrations of the insulin-like growth factors (IGF-I and IGF-II) were investigated in 235 blood samples taken from 145 healthy beef or dairy calves, bulls and cows of different breeds and ages. Autoradiography of Western ligand blots indicated different IGF binding protein (IGFBP) profiles between sera from different categories of cattle. Each IGF radioimmunoassay was validated by determining the effects of IGFBPs, ligand and contraligand, as well as serial dilution and comparison with results obtained after molecular sieve chromatography in acid. In female cattle mean values for IGF-I varied from 5.1 nmol/l in postparturient Holstein cows to 18.5-20.5 nmol/l in growing beef heifers, while mean IGF-II concentrations ranged from 30.0 nmol/l in the cows to 14.7-15.7 nmol/l in the beef heifer calves. In male cattle mean serum IGF-I ranged widely from 8.2 nmol/l in 1-day-old Holstein calves to 67.4 nmol/l in 16-month-old Simmental-type bulls. Mean IGF-II concentrations decreased from 22.9 nmol/l in 1-day-old Holstein bull calves to 11.9 nmol/l in 12-month-old beef bulls. Thus, total molar IGF concentrations were fairly stable in female cattle (24.7-35.1 nmol/l) but extended from 27.3 nmol/l to 81.8 nmol/l in the male cattle. The tendency for a reciprocal relationship between serum concentrations of these growth factors was most obvious in the periparturient cows.     

Immunolocalization of steroidogenic enzymes in the corpus luteum and placenta of the Japanese Shiba goat

In this study, we performed immunohistochemistry of cholesterol side-chain cleavage cytochrome P450 (P450scc), 3beta-hydroxysteroid dehydrogenase (3betaHSD), cytochrome 17alpha-hydroxylase P450 (P450c17), and cytochrome P450 aromatase (P450arom) in the corpus luteum and placenta of Shiba goats. The aim was to clarify the steroidogenic capability of the corpus luteum and placenta of Shiba goats. Ovaries containing corpora lutea were obtained from four adult Shiba goats during the luteal phase (day10; n=2) and pregnancy (90 and 120 days of gestation). Placenta was obtained from one Shiba goat on day 120 of gestation. The sections of the ovaries and placentae were immunostained using the avidin-biotin-peroxidase complex method (ABC) with polyclonal antibodies generated against steroidogenic enzymes of mammalian origin. All luteal cells expressed P450scc, 3betaHSD, P450c17 and P450arom. The distribution of P450scc, 3betaHSD, P450c17 and P450arom were not different during the luteal phase and pregnancy. P450arom showed a weak positive staining in late pregnancy (120 days). In addition, immunoreactions for P450c17 and P450arom were observed in syncytiotrophoblast of the placenta of one Shiba goat. These results indicate that, in Shiba goats, corpus luteum is not only an important source of progesterone but also has the ability to synthesize androgen and estrogen during the luteal phase and pregnancy. Also the placenta has the ability to synthesize androgen and estrogen in late pregnancy.     

Immunolocalization of steroidogenic enzymes in equine fetal adrenal glands during mid-late gestation

To elucidate the relationship between steroidogenic hormones and developing adrenal glands, we investigated the immunolocalization of steroidogenic enzymes in equine fetal adrenal glands during mid-late gestation. Fetal adrenal glands were obtained from three horses at 217, 225 and 235 days of gestation. Steroidogenic enzymes were immunolocalized using polyclonal antisera raised against bovine adrenal cholesterol side-chain cleavage cytochrome P450 (P450scc), human placental 3beta-hydroxysteroid dehydrogenase (3betaHSD), porcine testicular 17alpha-hydroxylase cytochrome P450 (P450c17) and human placental aromatase cytochrome P450 (P450arom). Histologically, cortex and medulla cells were clearly observed in the three fetal adrenal gland tissue samples. P450scc and P450c17 were identified in cortex cells close to medulla cells and in some medulla cells in the fetal adrenal glands. P450arom was present in both cortex and medulla cells in the fetal adrenal glands. However, 3betaHSD was not found in any of the equine fetal adrenal gland tissue samples. These results suggest that equine fetal adrenal glands have the ability to synthesize androgen and estrogen, which may play an important physiological role in the development of equine fetal adrenal glands.     

Long-term growth hormone-releasing factor administration on growth hormone, insulin-like growth factor-I concentrations, and bone healing in the Beagle.

Twelve 11 month old male Beagles were assigned to two treatment groups: a control group (saline) and a group receiving human growth hormone (GH)-releasing factor (hGRF) [1-29]NH2 (25 micrograms/kg, SC, TID). Treatment was started 6 days prior to surgery (day 1) and continued until necropsy (3 dogs per group/day) on d 29 or 58. Two porous polyethylene rods were surgically implanted on the lateral diaphysis of the femoral shaft and a 3 mm hole was drilled through the cortex between the two implants of each dog on day 1. Blood and urine were collected on d -6, 27 and 56. Human GRF injections produced a significant (P < 0.05) increase in GH release following each injection. An increase in GH response was also observed (P < 0.05) over time. The concentration of insulin-like growth factor-1 (IGF-1) increased for 5 weeks and then reached a plateau. None of the hematologic or urine measured parameters was affected by the treatment (P > 0.05). Albumin, calcium, and protein concentrations were higher (P < 0.05) on d 27 and 56 in GRF-treated animals. Histological sections of the onlay sites showed that bony ingrowth tended to be greater into the porous polyethylene material in GRF-treated animals than the controls at d 28 and 57, while no difference was observed in the degree of periosteal bone formation around the implants at either time period (P > 0.05). Bone formation into the cortical defect was greater in the GRF-treated dogs when compared to controls at day 57 only. In conclusion, chronic hGRF [1-29]NH2 treatment in Beagle dogs produced an increased GH response over time and increased IGF-1 concentrations. It also appeared to promote bony ingrowth into a porous polyethylene onlay and into a bony deficit.     

Expression of messenger ribonucleic acid encoding for steroidogenic acute regulatory protein and enzymes, and luteinizing hormone receptor during the spring transitional season in equine follicles

The period of spring transition, from the anovulatory to the ovulatory season, is characterized in many mares by cyclical growth and regression of large dominant follicles. These follicles produce only low concentrations of estradiol and it is thought that acquisition of steroidogenic competence by large follicles during spring transition is prerequisite in stimulating LH prior to first ovulation. In situ hybridization was used to localize and quantify expression of factors that play a key role in follicular steroidogenesis: StAR, P450scc (CYP11A1), P450c17 (CYP17), P450arom (CYP19), and LH receptor (LHr). One ovary was obtained from mares on the day after detection of an actively growing 30 mm transitional anovulatory follicle (defined as the transitional follicle), and the remaining ovary was removed at the third estrus of the breeding season on the day after the preovulatory follicle reached 30 mm in diameter (defined as the preovulatory follicle). Messenger RNAs encoding StAR, CYP11A1, and CYP17 were detected only in theca cells and CYP19 mRNA was confined to the granulosa layer. There was significantly lower expression of mRNAs for the steroidogenic enzymes, StAR (P<0.001) and LHr (P<0.05) in transitional follicles than in preovulatory follicles. In conclusion, large equine follicles during spring transition have low levels of mRNA encoding steroidogenic enzymes, StAR and LHr which will contribute to the steroidogenic incompetence of dominant follicles during spring transition and their subsequent regression.     

Cloning and expression of equine insulin-like growth factor binding proteins in normal equine tendon

OBJECTIVES: To define a portion of the nucleotide sequences of each of the 6 insulin-like growth factor (IGF) binding proteins (IGFBPs) in horses and describe patterns of messenger RNA (mRNA) and protein expression for IGFBPs in normal equine tendons. ANIMALS: 7 horses. PROCEDURE: Total RNA was extracted from the tensile region of normal superficial digital flexor tendons and reverse transcribed into complimentary DNA (cDNA). The cDNA was amplified via PCR, and products representing portions of each IGFBP were cloned and sequenced. Nucleotide sequences were used to deduce the amino acid sequences, and both nucleotide and predicted amino acid sequences were compared with those published for bovine, human, mouse, and ovine IGFBPs. Gene expression was quantitated by real-time PCR assay, and protein expression was evaluated by western ligand blot (WLB). RESULTS: Clones ranged in size from 262 to 522 bp and had high degrees of sequence homology with other mammalian species. Sequence homology was highest between bovine and equine IGFBPs (86% to 95%) and amongst the IGFBP-5 sequences from the various species (92% to 95%). Message for IGFBP-2 to -6, but not IGFBP-1, was expressed in normal tendon. Protein expression for IGFBP-2, -3, and -4 was detected byWLB in normal tendon and markedly increased in damaged tendons. CONCLUSIONS AND CLINICAL RELEVANCE: Results provide basic information and tools needed for further characterization of the role of the IGF system in tendon healing and may lead to the ability to potentiate the response of healing tendon to exogenous IGF-I via concurrent manipulation of IGFBPs.     

Changes in circulating insulin-like growth factor-I, insulin-like growth factor binding proteins, and leptin in weaned pigs infected with Salmonella enterica serovar Typhimurium

One of the hallmarks of the pathophysiology of enteric disease in young pigs is reduced growth performance. This reduction in growth is associated with changes in the endocrine somatotropic growth axis. Our laboratory previously demonstrated that circulating insulin-like growth factor-I (IGF-I) was reduced in pigs infected with Salmonella enterica serovar Typhimurium (S. typhimurium) while circulating growth hormone remained unchanged. The objective of the current study was to determine if infection with S. typhimurium also was associated with changes in circulating IGF binding proteins (IGFBP). In addition, pigs experiencing active enteric disease have reduced feed intake. Because this inappetence may be related to systemic appetite reduction signals, we also evaluated circulating leptin in pigs undergoing active S. typhimurium-induced enteric disease. Crossbred pigs were penned in environmentally controlled rooms with free access to feed and water. Following an acclimation period, pigs were gavaged with 10(10) cfu of S. typhimurium (SAL; n=6) or were given a similar volume of sterile growth media (CON; n=6). Rectal temperatures and feed intakes were measured daily through 168 h to track the time course of the response to S. typhimurium infection. Samples of serum were obtained by jugular venipuncture at 0, 24, 48, 96 and 168 h after infection. Sera were frozen until evaluation for IGF-I by immunoradiometric assay (IRMA). In addition, sera were subjected to western ligand blotting utilizing 125I-IGF-I and 125I-IGF-II. Images were evaluated for total IGFBP and IGFBP-3 by densitometric analyses. Rectal temperature was increased in SAL pigs 24h post-infection (P<0.001) but not at other times. Feed intake was reduced in SAL pigs during the intervals 24-72 h (P<0.001) and 96-144 h (P<0.05) after infection. Serum IGF-I, expressed as a percentage of the 0 h concentration, was reduced in SAL pigs versus CON pigs at 48 h (28.1+/-18.7% versus 102.2+/-17.1%; P<0.01) and 96 h (20.0+/-18.7% versus 128.4+/-17.0%; P<0.0001) post-infection. Both total IGFBP and IGFBP-3, as estimated by ligand blotting, also were reduced in infected pigs at 48 h postchallenge (P<0.05). IGFBPs were similar between the two treatments at other sampling times. Concentrations of IGFBP-3 also were estimated utilizing an IRMA for human IGFBP-3. Serum IGFBP-3 was reduced in S. typhimurium-infected pigs at 24 h (P<0.01), 48 h (P<0.001), 96 h (P<0.001), and 168 h (P<0.05). Serum leptin levels were similar between SAL and CON pigs. The data suggest that swine enteric disease is associated with reduced circulating IGF-I and reductions in total IGFBP and IGFBP-3. However, serum leptin was not affected by enteric disease challenge.     

Plasma insulin-like growth factor-I concentrations increase during the estrous phase in goats

The objective of this study was to characterize plasma insulin-like growth factor-I (IGF-I) profiles during the estrous cycle in goats. Frequent blood samples were drawn during the day of estrus and during the luteal phase on Day 10 after estrus, and plasma growth hormone (GH) and IGF-I profiles were examined. Then, daily blood samples were drawn throughout the estrous cycle or during induction of estrus by prostaglandin F(2alpha) (PGF(2alpha)) to further clarify the IGF-I profiles. GH was secreted in an episodic manner in the estrous and luteal phases in goats. There were no significant differences in the mean concentrations, pulse amplitude and pulse frequency of GH between the estrous and luteal phases. IGF-I concentrations during estrous phase were higher than those in the luteal phase (P<0.05). Plasma IGF-I increased approximately two days before behavioral estrus, and the IGF-I peak was observed in accordance with the appearance of estrus. The elevated IGF-I levels then declined to basal values 4 to 5 days after estrus. When estrus was induced by PGF(2alpha), plasma IGF-I concentrations increased after treatment, and the concentration 2 days after treatment (day of appearance of behavioral estrus) was significantly higher than concentrations before treatment (P<0.05). The elevated IGF-I levels then declined during the 3 days after treatment. These results indicate that plasma IGF-I concentrations increase during estrus in goats.     

Increased degradation of insulin-like growth factor-I in serum from feed-deprived steers

Severe feed restriction decreases serum insulin-like growth factor I (IGF-I) concentration in animals, and this decrease is thought to be due to reduced IGF-I production in the liver. The objective of this study was to determine whether feed deprivation also increases degradation of serum IGF-I and serum levels of IGF binding protein 3 (IGFBP-3) and acid-labile subunit (ALS), which inhibit IGF-I degradation and increase IGF-I retention in the blood by forming a ternary complex with IGF-I, in cattle. Five steers had free access to pasture, and another five were deprived of feed for 60 h. Serum concentration of IGF-I and liver abundance of IGF-I mRNA at the end of the 60-h period were 50% and 80% lower, respectively, in feed-deprived steers than in fed steers. Less ¹²⁵I-labeled IGF-I remained intact after a 45-h incubation in sera of feed-deprived steers than in sera of fed steers, suggesting that serum IGF-I is more quickly degraded in feed-deprived animals. Serum levels of IGFBP-3 and ALS were decreased by 40% and 30%, respectively, in feed-deprived steers compared with fed steers. These decreases were associated with more than 50% reductions in IGFBP-3 and ALS mRNA in the liver, the major source of serum IGFBP-3 and ALS. Taken together, these results suggest that feed deprivation reduces serum concentration of IGF-I in cattle not only by decreasing IGF-I gene expression in the liver, but also by increasing IGF-I degradation and reducing IGF-I retention in the blood through decreasing IGFBP-3 and ALS production in the liver.     

Plasma concentrations of growth hormone and insulin-like growth factor-I in prepuberal quarter horses and ponies

The hypotheses were tested that among types of horses with phenotypically different mature sizes, a difference in pattern of secretion of 1) GH and 2) insulin-like growth factor-I (IGF-I) would exist prepuberally. To test these hypotheses, plasma was collected each 20 min for 8 hr from three types of horses [Quarter Horses (n=5), ponies (n=4), and Quarter Horse-pony F₁ crosses (n=5)] at 2, 4, and 10 months of age. Plasma concentrations of GH and IGF-I were determined by RIA and the patterns of secretion were quantified. Type of horse had no effect on tonic patterns of secretion of GH (P=0.92) or IGF-I (P=0.39), so the hypotheses were rejected and the data were pooled across types within age. Mean plasma concentrations of GH did not differ (P=0.74) with respect to age of horse. In contrast, number of pulses of GH per 8 hour (2 months = 2.3±0.4; 4 months = 2.2±0.5; 10 months = 2.8±0.9) and the interval between pulses (2 months = 87.1±23.1; 4 months = 121.7±25; 10 months = 111.5±15 min) changed quadratically (P=0.03 and P=0.02). Plasma concentrations of IGF-I decreased quadratically (P=0.01) from 2 months through 10 months of age. These data provide evidence to suggest that tonic secretion of GH and IGF-I may differ among prepuberal Quarter Horses and ponies with respect to age of horse but not type of horse.     

Effects of flax supplementation and a combined trenbolone acetate and estradiol implant on circulating insulin-like growth factor-I and muscle insulin-like growth factor-I messenger RNA levels in beef cattle

We evaluated effects of a 5% (dry matter basis) ground flaxseed supplement (flax) and a trenbolone acetate and estradiol-17beta implant, Revalor-S, on circulating IGF-I and muscle IGF-I messenger RNA (mRNA). Sixteen crossbred yearling steers (initial BW = 397 kg) were assigned randomly to one of four treatments: 1) flax/implant; 2) nonflax/implant; 3) flax/nonimplant; and 4) nonflax/nonimplant. Serum was harvested from blood collected on d 0 (before implant or flax addition), 14, and 28, and used in subsequent analyses of circulating IGF-I. Biopsy samples (0.5 g) were obtained from the longissimus muscle on d 0, 14, and 28. Total RNA was isolated from the muscle samples, and real-time quantitative-PCR was used to assess relative differences in IGF-I mRNA. Flax supplementation had no effect (P > 0.10) on circulating IGF-I concentrations. Following implantation, sera from implanted steers had 52 and 84% greater (P < 0.05) IGF-I concentrations than sera from nonimplanted steers on d 14 and 28, respectively. On d 28, local muscle IGF-I mRNA levels increased 2.4-fold (P < 0.01) in biopsy samples obtained from implanted compared with nonimplanted steers. Muscle biopsy samples from nonflax cattle had 4.4-fold higher (P < 0.01) levels of IGF-I mRNA than those from flax cattle on d 28. To determine whether a component of flax, alpha-linolenic acid (alphaLA), was directly responsible for IGF-I mRNA down-regulation, we incubated primary cultures of bovine satellite cells, from implanted and nonimplanted steers, in two concentrations of alphaLA (10 nM and 1 microM). An implant x dose interaction (P < 0.05) was observed for IGF-I mRNA concentrations in bovine satellite cells cultured for 72 h with alphaLA. Satellite cells from nonimplanted steers had similar (P > 0.10) IGF-I mRNA concentration regardless of the level of alphaLA exposure; however, satellite cells from implanted steers exposed to 10 nM and 1 microM alphaLA had 2.5- and 2.0-fold greater IGF-I mRNA levels, respectively, than cells from implanted steers that were not exposed to alphaLA (P < 0.05). Administration of a Revalor-S implant increased circulating IGF-I and local muscle IGF-I mRNA concentrations in finishing cattle. However, muscle IGF-I mRNA levels were decreased by flax supplementation. Muscle cell culture experiments suggested that alphaLA was not responsible for the IGF-I mRNA down-regulation.