Estrogen and androgen elicit growth hormone release via dissimilar patterns of hypothalamic neuropeptide secretion.

The dimorphic pattern of growth hormone (GH) secretion and somatic growth in male and female mammals is attributable to the gonadal steroids. Whether these hormones mediate their effects solely on hypothalamic neurons, on somatotropes or on both to evoke the gender-specific GH secretory patterns has not been fully elucidated. The purpose of this study was to determine the effects of 17beta-estradiol, testosterone and its metabolites on release of GH, GH-releasing hormone (GHRH) and somatostatin (SRIF) from bovine anterior pituitary cells and hypothalamic slices in an in vitro perifusion system. Physiological concentrations of testosterone and estradiol perifused directly to anterior pituitary cells did not affect GH releases; whereas, dihydrotestosterone and 5alpha-androstane-3alpha, 17beta-diol increased GH. Perifusion of testosterone at a pulsatile rate, and its metabolites and estradiol at a constant rate to hypothalamic slices in series with anterior pituitary cells increased GH release. The androgenic hormones increased GHRH and SRIF release from hypothalamus; whereas, estradiol increased GHRH but decreased SRIF release. Our data show that estradiol and the androgens generated distinctly different patterns of GHRH and SRIF release, which in turn established gender-specific GH patterns.

Composition, solubility and gel properties of salt soluble proteins from two bovine muscle types.

The composition, pH solubility profile and thermal gelation behavior of two bovine muscles, vastus intermedius (VI, predominately red fibers) and semimembranosus (SM, predominantly white fibers) were compared. VI had a higher fat content and pH and lower protein content than SM. Between pH 5.2 and 5.8, the salt soluble proteins (SSP) from SM were more soluble than those from VI at the same pH, whereas solubilities above pH 6.0 were similar. Properties of SSP gels were measured at pH 5.5 and 6.1, the ultimate pH for SM and VI, respectively. Water lost from the VI gels due to syneresis was about 3 times greater than that lost from SM gels. VI gels prepared at pH 5.5 were firmer (p<0.05) than at pH 6.1, whereas deformability of SM gels at pH 6.1 were greater (p<0.05) than at pH 5.5. No differences (p>0.05) were observed between the firmness or deformability of VI or SM gels when compared at the same pH. Results suggest that ultimate muscle pH and fiber type do influence the properties of bovine SSP gels, although the effect is not as great as that previously reported for poultry muscle proteins.

Increases in insulin-like growth factor binding protein-2 accompany decreases in proliferation and differentiation when porcine muscle satellite cells undergo multiple passages.

Subjecting cloned porcine myogenic satellite cells to multiple passages leads to decreased rates of cell division and myotube formation. Because IGF have been implicated in the regulation of muscle cell proliferation and differentiation, the present study was conducted to characterize secretion of IGF-I and IGF-binding proteins (IGFBP) in cultures of cloned porcine satellite cells at two stages of multiple passaging. To this end, we obtained a single porcine satellite cell clone that demonstrated relatively high capacities for cellular proliferation and differentiation into myotubes at the fifth passage but that had greatly diminished capacities for proliferation and myotube formation by the seventh passage. The predominant IGFBP secreted by this satellite cell clone was immunologically identified as IGFBP-2, and quantities of it were increased in medium from seventh-passage cultures. Quantities of IGF-I in medium were determined with a newly developed "titration" radioimmunoassay in which interference from IGFBP was minimized by adding a range of saturating quantities of IGF-II. Medium IGF-I concentrations in seventh-passage cultures were also increased relative to the fifth-passage cultures when expressed per unit of DNA. It is hypothesized that the observed increase of IGF-I in medium likely resulted from protective sequestration of IGF-I by IGFBP-2 rather than from enhanced IGF-I secretion. In summary, these data suggest that multiple passaging of cloned porcine satellite cells results in increased secretion of IGFBP-2, which is associated with depressed cell proliferation and myotube formation, perhaps because the increased IGFBP-2 sequestered IGF-I and reduced its bioactivity.

Testosterone up-regulates androgen receptors and decreases differentiation of porcine myogenic satellite cells in vitro.

Accumulation of DNA is essential for muscle growth, yet mechanisms of androgen-induced DNA accretion in skeletal muscle are unclear. The purpose of this study was to determine whether androgen receptors (AR) are present in cultured skeletal muscle satellite cells and myotubes and examine the effects of testosterone on satellite cell proliferation and differentiation. Immunoblot analysis using polyclonal AR antibodies (PG-21) revealed an immunoreactive AR protein of approximately 107 kDa in porcine satellite cells and myotubes. Immunocytochemical AR staining was confined to the nuclei of satellite cells, myotubes, and muscle-derived fibroblasts. Administration of 10(-7) M testosterone to satellite cells, myotubes, and muscle-derived fibroblasts increased immunoreactive AR. In satellite cells and myotubes, AR increased incrementally after 6, 12, and 24 h of exposure to testosterone. Testosterone (10(-10) - 10(-6) M), alone or in combination with insulin-like growth factor I, basic fibroblast growth factor, or platelet-derived growth factor-BB, had no effect (P > 0.01) on porcine satellite cell proliferation, and testosterone pretreatment for 24 h did not alter the subsequent responsiveness of cells to these growth factors. Satellite cell differentiation was depressed (20-30%) on days 2-4 of treatment with 10(-7) M testosterone. This effect was not reversible within 48 h after treatment withdrawal and replacement with control medium. These data indicate that satellite cells are direct targets for androgen action, and testosterone administration increases immunoreactive AR protein and reduces differentiation of porcine satellite cells in vitro.

Perifusion model system to culture bovine hypothalamic slices in series with dispersed anterior pituitary cells.

Dispersed bovine anterior pituitary cells were incubated either in static or perifusion cultures to assess basal growth hormone release as well as stimulatory and inhibitory effects of growth hormone-releasing hormone and somatostatin, respectively, on growth hormone release. Total concentrations of growth hormones over a 12-hour incubation period were fivefold greater in perifused than in static cultures (2034 +/- 160 vs. 387 +/- 33 ng/12 h). A dose-dependent increase in growth hormone secretion in response to challenge with growth hormone-releasing hormone (10(-12) to 10(-8) M) for 1 h was observed in both static and perifusion cultures; however, perifused cells were more responsive to the same concentration of neuropeptide than those in static culture. Concentrations of somatostatin (10(-12) to 10(-8) M) for 1 h did not inhibit basal growth hormone secretion in either static or perifusion cultures. To establish the hypothalamic-pituitary in series-perifusion model, slices of the hypothalamus, immediately adjacent to the sagittal midline, were perifused in series with anterior pituitary cells, and media effluent was assayed for growth hormone concentrations. Release of growth hormone was pulsatile and seemed to mimic the episodic pattern of bovine secretion. Hypothalamic slices were placed in one chamber of the perifusion system, and basal secretion of growth hormone-releasing hormone and somatostatin was pulsatile in media effluent. Tissue viability of hypothalamic slices and anterior pituitary cells was evaluated by KCl depolarization. Tissues were viable for at least 120 h. Thus, this hypothalamo-pituitary dual chamber perifusion system is a valid in vitro model to study regulation of growth hormone secretion.

RNA transcription in porcine skeletal muscle nuclei during postnatal development.

Postnatal developmental pretranslational regulation of skeletal muscle alpha-actin gene expression was investigated. Northern blot analysis of skeletal muscle alpha-actin and beta-tubulin mRNA from 1- and 28-day-old pigs indicated that there are developmental increases in alpha-actin mRNA abundance (P < 0.03) and no significant changes in beta-tubulin mRNA (P > 0.1). A system for isolation of nuclei from porcine skeletal muscle and for transcriptional "run-on" analysis was established in order to investigate the regulatory mechanism of developmental changes in porcine skeletal muscle protein. Skeletal muscle nuclei were isolated from longissimus dorsi (LD) muscle of 1- and 28-day-old pigs by adapting a method to isolate nuclei from cardiac muscle. Results from a [3H]-UTP incorporation assay indicate that these nuclei preparations have the capacity to synthesize RNA and attain maximum incorporation after 40-45 min at 26 degrees C. Messenger RNA syntheses from skeletal muscle nuclei from 1- and 28-day-old pigs were not significantly different (P > 0.25). All nascent tRNA, rRNA, and mRNA in the nuclei were elongated since [3H]-UTP incorporation was reduced after addition of 0.05 micrograms/ml alpha-amanitin to the transcription mixture. Transcription "run-on" assay results indicated that more (P < 0.02) skeletal muscle alpha-actin pre-mRNA was synthesized in the 28-day-old pig skeletal muscle nuclei than in the 1-day-old pig skeletal muscle nuclei. These results indicate that the relative increase in skeletal muscle alpha-actin mRNA observed in the older animals was due, at least in part, to an increase in the transcriptional activity of the skeletal muscle alpha-actin gene.

Skeletal muscle growth and protein turnover in neonatal boars and barrows.

The objective of this study was to determine the effect of castration, within 24 h after birth, on skeletal muscle growth and protein metabolism in neonatal pigs at 1, 2, and 4 wk of age. Four additional pigs were slaughtered at birth to obtain initial body composition. All other pigs were infused with [14C]tyrosine for 6 h before slaughter to determine in vivo fractional protein synthesis rates (FSR). At slaughter, muscle bundles were removed from the semitendinosus and incubated with [3H]tyrosine to determine in vitro protein synthesis rates. Nucleic acids and protein were determined on the semitendinosus muscle. Testosterone concentrations, determined at weekly intervals, peaked in boars at 3 wk of age. Castration at birth did not affect combined weights of the semitendinosus, longissimus, triceps brachii, and brachialis muscles. Likewise, neither in vitro protein synthesis rates nor in vivo FSR were affected by castration. However, a developmental decline in in vivo FSR and in vitro protein synthesis rates occurred from 1 to 4 wk. Neither concentrations nor total quantity of protein, RNA, or DNA in the semitendinosus muscle differed between neonatal boars and barrows at any age. Concentrations of DNA and RNA at 4 wk were two- and threefold lower, respectively, than at birth. Protein:DNA and protein:RNA ratios increased three- and sixfold, respectively, from birth to 4 wk. Testosterone concentrations had little effect on skeletal muscle growth and protein turnover rates during this neonatal period.

Skeletal muscle growth and expression of skeletal muscle alpha-actin mRNA and insulin-like growth factor I mRNA in pigs during feeding and withdrawal of ractopamine.

Sixty crossbred barrows were used to study the effect of ractopamine (a phenethanolamine/beta-adrenergic agonist) treatment and its withdrawal on muscle growth and on the relative abundance of skeletal muscle alpha-actin (sk-alpha-actin) mRNA and of liver and longissimus muscle IGF-I mRNA at 4 wk. Ractopamine was fed (20 ppm) for periods of 2, 4, and 6 wk (six pigs per group). Additional pigs (four per group) were fed ractopamine (20 ppm) for 6 wk and then slaughtered 1, 3, and 7 d after withdrawal of ractopamine. Ractopamine increased (P < .05) longisimus muscle weight and protein content, although protein concentrations were not different. The increased muscle weight and protein content attained by feeding ractopamine for 6 wk was retained when ractopamine was withdrawn. The RNA and DNA concentrations did not change, whereas total DNA and RNA content per muscle was 18 and 26.7% greater, respectively, in ractopamine-treated pigs at 4 wk, but there were no differences at 2 or 6 wk or among the withdrawal groups. The relative abundance of sk-alpha-actin mRNA in the longissimus muscle was 41 and 62% greater (P < .05) in treated animals at 2 and 4 wk but was similar to that in controls at 6 wk and during the withdrawal period. The relative abundance of IGF-I mRNA in liver and longissimus muscle was not altered with ractopamine treatment for 4 wk. These results indicate that the ractopamine-enhanced muscle growth may result from increased myofibrillar gene expression at the pretranslational level, which is maximal with short-term treatment of ractopamine.(ABSTRACT TRUNCATED AT 250 WORDS)

Transforming growth factor-beta, basic fibroblast growth factor, and platelet-derived growth factor-BB interact to affect proliferation of clonally derived porcine satellite cells.

Transforming growth factor beta-1 (TGF-beta) stimulated porcine satellite cell proliferation in basal serum-free medium by 25%, but inhibited growth in serum-containing medium by 58%. The effect of TGF-beta on cell proliferation in serum-free medium was examined in combination with the following human recombinant growth factors: platelet-derived growth factor-BB (PDGF), basic fibroblast growth factor (FGF), insulin-like growth factor I (IGF-I), and epidermal growth factor (EGF). TGF-beta inhibited PDGF-stimulated proliferation, enhanced FGF-stimulated proliferation, and had no effect on proliferation stimulated by IGF-I. The response of satellite cells to EGF and TGF-beta in serum-free medium was not different than TGF-beta alone. TGF-beta depressed proliferation stimulated by the following combinations of two growth factors: PDGF and IGF-I, PDGF and EGF, PDGF and FGF, and IGF-I and EGF. In combination with IGF-I and FGF, TGF-beta did not affect proliferation. TGF-beta inhibited proliferation stimulated by the combination of PDGF, EGF, and IGF-I, but had no effect on proliferation stimulated by combinations of three growth factors that included FGF. FGF stimulated proliferation in Minimum Essential Medium containing 10% porcine serum (MEM-10% PS) by 13% above control. When the combination of TGF-beta and FGF was added to MEM-10% PS, a 78% increase in proliferation was observed. Polyclonal antihuman PDGF-AB (this form neutralizes PDGF-AA, AB, and BB) reduced proliferation in MEM-10% PS by 44%. The combination of TGF-beta and anti-PDGF-AB reduced proliferation by 59%, indicating the effects were not additive. These data indicate that: (1) FGF and TGF-beta interact to increase proliferation of clonally derived porcine satellite cells, and (2) the inhibitory effect of TGF-beta on proliferation of clonally derived porcine satellite cells can be primarily attributed to a reduction in the mitogenic effects of PDGF.

Fibroblast growth factor, epidermal growth factor, insulin-like growth factors, and platelet-derived growth factor-BB stimulate proliferation of clonally derived porcine myogenic satellite cells.

Clonally derived cultures of porcine skeletal muscle satellite cells were developed. The mitogenic effects of basic fibroblast growth factor (bFGF), epidermal growth factor (EGF), insulin-like growth factors (IGF-I and -II), and platelet-derived growth factors (PDGF-AA and -BB) were examined. Individually, bFGF, IGFs, and PDGF-BB stimulated proliferation of porcine satellite cells grown in basal serum-free medium or Minimum Essential Medium containing 2% fetal bovine serum (MEM-2% FBS). EGF stimulated proliferation in MEM-2% FBS, but neither EGF nor PDGF-AA were mitogenic when added to serum-free medium. The interactions among bFGF, EGF, IGF-I, and PDGF-BB were examined in serum-free medium, using growth factor concentrations shown in dose-response experiments to induce maximal proliferative responses (10 ng/ml bFGF, EGF and PDGF-BB, and 50 ng/ml IGF-I). The combination of bFGF and IGF-I dramatically increased proliferation, and IGF-I also synergized with EGF to increase proliferation. EGF, IGF-I, and bFGF interacted with PDGF-BB to stimulate proliferation. With the exception of EGF and bFGF, combinations of two growth factors typically resulted in greater than additive responses. Simultaneous exposure of satellite cells to bFGF, PDGF-BB, EGF, and IGF-I produced a fivefold increase in DNA compared to cells grown in basal serum-free medium. Elimination of EGF did not reduce the mitogenic response, yet removal of IGF-I, bFGF, or PDGF-BB reduced proliferation by approximately 40, 20, and 10%, respectively. These mitogens are likely physiological regulators of porcine satellite cell activity.

Fatty acid profiles, lipogenesis, and lipolysis in lipid depots in finishing pigs treated with recombinant porcine somatotropin.

Thirty-two pigs (average weight, 74 kg) were used to determine the effects of recombinant porcine somatotropin (rpST) and dietary CP concentration on carcass and noncarcass tissues. Pigs were injected intramuscularly with either rpST (50 micrograms.kg BW-1.d-1; n = 16) or vehicle (n = 16) at 0900 for 24 d. Half the treated and control pigs were given ad libitum access to either a 14 or 20% CP corn-soybean meal diet. The left side of the carcass was physically dissected into separable skin, soft tissues, and bone. Tissue samples were obtained for enzyme assays, proximate analysis, and fatty acid profiles. Proximate analysis (fat, water, protein) of adipose tissue (AT) samples indicated that rpST decreased the percentage of ether extractable lipid (P < .01) and increased the percentage of protein and water (P < .05, P < .01) in intermuscular (IM), subcutaneous (SC), and intrafascicular (IF) AT depots and slightly altered fatty acid profiles of longissimus muscle IF and SC AT. Fatty acid synthesis and malic enzyme activity were decreased by rpST (P < .01), suggesting that lipogenesis was decreased; however, lipolysis was unaffected. Pigs fed the high-CP diet (20%) had decreased AT malic enzyme activity (P < .05) and fatty acid synthesis (P < .05). Additionally, pigs fed the high-CP diet had greater kidney weights (P < .01). Heart, liver, and kidney weights were heavier (P < .01) in pigs treated with rpST, whereas skin and total bone weight were unaffected. Neither weights nor lengths of four individual long bones were affected by dietary protein concentration or administration of rpST.

Alpha-2 adrenergic receptor activity in porcine adipocytes is androgen and age/cell size dependent.

1. Using epinephrine plus propranolol we demonstrated alpha-2 adrenergic receptor (alpha 2AR) activity (49% inhibition of theophylline stimulated lipolysis) in adipocytes of adult intact male pigs (61 kg body wt and 97 microns adipocyte diameter). 2. Dose titration with an alpha 2AR antagonist (yohimbine) confirmed alpha 2AR associated activity. 3. No alpha 2AR activity was observed in younger male or castrated male pigs. 4. The inhibitory action on lipolysis via the alpha 2AR in pigs is dependent on androgen status and adipocyte size or age.

Determination of the relative abundance of skeletal muscle alpha actin mRNA in muscle of livestock species.

Three market-weight animals of meat-producing livestock species were slaughtered to obtain porcine (barrows), bovine (steers), ovine (wethers), and avian (cockerels) tissue samples. The four tissues of interest were skeletal muscle, heart, smooth muscle (stomach or gizzard), and liver. Total RNA was isolated from each tissue and then hybridized to a human skeletal (sk)-alpha-actin [32P]cDNA probe using both dot blot and Northern blot hybridization. No hybridization was observed with RNA from liver or smooth muscle from any of the species, suggesting little or no hybridization to nonmuscle and smooth muscle beta- and gamma-actin isoforms. The human sk-alpha-actin probe hybridized to RNA from skeletal muscle of pigs, cattle, sheep, and chickens, although relative hybridization was 75% less with chicken RNA. The hybridization was limited specifically to a band at 1.6 kb (kilobases), the known length of sk-alpha-actin mRNA. Hybridization was observed with RNA from pig heart (1.6 kb) and the relative abundance was consistently 7 to 10% of that observed with porcine skeletal muscle, even as stringency conditions were increased. These results indicate that the human sk-alpha-actin probe can be used to determine alpha-actin mRNA expression in skeletal muscle for pigs, cattle, and sheep.

Adipogenic cell line TA1: a suitable model to study the effect of beta-adrenergic agonists on lipid metabolism.

The stable adipogenic cell line TA1 was investigated as a potential in vitro system to examine effects of beta-adrenergic agonists on lipid metabolism at the cellular level. Initial experiments were conducted to establish whether dexamethasone, indomethacin, or both in combination induce rapid differentiation of TA1 preadipocytes to adipocytes. Based on activity of fatty acid synthase, dexamethasone and indomethacin, individually and in combination, were observed to induce differentiation in TA1 cells at different rates (dexamethasone/indomethacin greater than indomethacin greater than dexamethasone). Dexamethasone/indomethacin induced complete differentiation in TA1 cells 4 days after confluence, as indicated by increased activity of fatty acid synthase, glycerol-3-phosphate dehydrogenase, and malic enzyme. Finally, mature TA1 adipocytes were treated with various concentrations of isoproterenol and ractopamine to determine the responsiveness of TA1 adipocytes to a beta-adrenergic challenge. Glycerol release was increased and fatty acid synthase activity was decreased in a dose-dependent manner for both isoproterenol and ractopamine. These results indicate that fully differentiated TA1 adipocytes may be useful to study direct cellular effects of lipolytic and lipogenic agents on lipid metabolism.

Skeletal muscle protein metabolism and serum growth hormone, insulin, and cortisol concentrations in growing steers implanted with estradiol-17 beta, trenbolone acetate, or estradiol-17 beta plus trenbolone acetate.

Skeletal muscle protein degradation, measured by urinary N tau-methylhistidine excretion, and circulating concentrations of growth hormone (GH), insulin (INS), and cortisol (CT) were monitored in steers before and after implantation with estradiol-17 beta (E2; 24 mg) and trenbolone acetate (TBA; 300 mg). Yearling crossbred steers (n = 43) were randomly assigned to four treatment groups in a 2 x 2 factorial arrangement: nonimplanted controls (C); TBA; E2; and TBA plus E2 (TBA+E2). A subgroup (Block 1) of 16 steers was bled on d -12, 31, and 72 after implanting. Deposition of skeletal muscle protein was markedly increased (P less than .001) by E2 and TBA+E2 treatment. This response occurred mainly within the first 40 d after implantation and declined (P less than .001) in concert with decreasing (P less than .01) concentration of serum E2. Anabolic steroid treatment did not affect the rate of skeletal muscle protein breakdown. There was no apparent relationship between reduced serum CT concentration (linear effect; P less than .01) in TBA-treated steers and skeletal muscle protein degradation rate. Blood concentration and pulse activity of INS were not affected by anabolic steroid administration. Both TBA- and TBA+E2-implanted steers displayed a linear decrease (P less than .05) in serum GH concentration over time, which was similar to C. Lowered mean GH concentration resulted from a reduction (TBA main effect; P less than .05) in pulse amplitude of GH. Unlike TBA, TBA+E2, and C, only E2 maintained serum GH concentrations over time. Although increased muscle protein deposition was evident in TBA+E2-treated steers, an obvious causal relationship between this response and circulating GH, INS, and CT was not revealed. These results do not support the concept that combined androgenic agent and estrogen administration effectively reduce bovine muscle protein degradation by static modulation of circulating endogenous anabolic and antianabolic hormones.

Androgens modulate growth hormone-releasing factor-induced GH release from bovine anterior pituitary cells in static culture.

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 (P > 0.05) 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 (P < 0.05) GH from 58 to 134 ng/ml. Incubation with E2 or 3 alpha-diol did not affect (P > 0.05) GRF-induced GH release; however, preincubation with T (10(-5) M) and DHT (10(-9), 10(-8) and 10(-7) M) increased (P < 0.05) 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.

Quantitative characterization of beta-adrenergic receptor subtypes in porcine adipocytes.

1. Two populations of beta-adrenergic receptor (beta AR) subtypes and their proportions were characterized in adipocytes isolated from subcutaneous adipose tissue of castrated male crossbred pigs (60-85 kg). 2. Specific binding of the radioligand 125I-iodopindolol (IPIN) to crude plasma membranes (70-90% of total binding) reached equilibrium conditions in 30 min (38 degrees C), was tissue concentration-dependent, stereospecific and saturable (bmax = 168 +/- 5.8 fmol/mg protein). 3. Displacement curves by ICI 89,406 were best-fit by a two site model (P less than 0.01) that indicated the presence of two receptor populations and selectivity of IPIN for the beta 2AR subtype. 4. Forty-five percent of the receptors had a high affinity for ICI 89,406, Ki = 2.27 +/- 0.68 nM and were classified as beta 1AR.

Conditions for isolation and culture of porcine myogenic satellite cells.

Myogenic satellite cells were isolated from semimembranosus muscles of 4-8 week-old pigs. Muscles were ground and incubated in 0.8 mg/ml Pronase solution for 40 min at 37 degrees C. Following enzymatic digestion, cells were separated from muscle debris by differential centrifugation and sequential filtering through 500 and 53 microns nylon mesh. Primary cultures grown in 16 mm diameter cell culture wells were used to evaluate five sera, media, and substrata for their ability to promote satellite cell proliferation and differentiation. Porcine satellite cell proliferation and myotube formation were optimized in cultures grown on gelatin-coated substratum in the presence of Minimum Essential Medium-alpha supplemented with 10% fetal bovine serum (FBS) (P less than 0.01). Maximum fusion was induced by 48 hr exposure to 2% FBS, horse serum, or lamb serum. These data 1) document the first evidence that myogenic satellite cells can be isolated from porcine skeletal muscle, and 2) identify culture conditions which optimize proliferation and myotube formation of porcine satellite cells.

Body composition of animals treated with partitioning agents: implications for human health.

Excess fat in meat products has been identified as a dietary problem by public health officials. The meat animal industry has responded during the last 25 years to concerns about excess fat intake from animal products by implementing strategies to depress fat deposition and increase lean (protein) tissue gain in meat animals. The most successful strategy to date is the use of large, late-maturing animals for meat production. At desired market weights, these animals are much leaner than smaller, early-maturing animals. In addition, exogenous agents such as anabolic steroids (FDA approved for cattle) have been used to increase lean gain and depress fat deposition in cattle. Growth hormone (GH) and beta-adrenergic agonists (beta AA) are not yet approved by the FDA, but if/when approved would also markedly increase lean gain and depress fat deposition. Both GH and beta AA are called partitioning agents because they partition nutrients and energy toward lean (protein) accretion and dramatically lower fat deposition. Contingent on approval by the FDA and subsequent adoption of partitioning agents by the animal industry would result in meat products containing less and 30% of total calories from fat.