Biomedical subjects

M E Doumit

Publications and source records attributed to M E Doumit.

18 recordsLinked to original sources

PubMed2006

Effects of halothane sensitivity on mobility status and blood metabolites of HAL-1843-normal pigs after rigorous handling.

The objective of this study was to determine if HAL-1843-normal pigs that respond abnormally to halothane anesthesia were more likely to become nonambulatory (NA) when subjected to rigorous handling than pigs that exhibit a normal response to halothane. After a 1,100-km transport, pigs exhibiting low (HS-L; n = 33), intermediate (HS-I; n = 10), and high (HS-H; n = 47) sensitivity to halothane were moved through a 36.6-m long aisle that was 2.1 m wide at each end and 0.6 m wide in the middle 18.3 m. Ten groups of 8 pigs were briskly moved down the aisle and back 4 times, receiving a minimum of 1 electrical prod per pass (8 prods/pig). Before testing, rectal temperature was measured, open-mouth breathing and skin discoloration were visually evaluated, and a blood sample was collected from each pig. After the test, the pigs were returned to their pens, and the same measurements were taken immediately posttest and 1 h posttest (no blood at 1 h posttest). Pigs that were HS-H were more prone to becoming NA compared with HS-L pigs (P < 0.02). Regardless of halothane status, a greater number of pigs exhibited open-mouth breathing and skin discolorations immediately posttest than at the pretest or 1 h posttest times (P < 0.05). No differences were observed in blood metabolites between the different halothane sensitivity categories. However, pigs that became NA had elevated blood levels of creatine phosphokinase, lactate, glycerol, nonesterified fatty acids, ammonia, and urea nitrogen before testing (P < 0.05). Collectively, these data suggest HS-H pigs are more susceptible to becoming NA than HS-L. The elevated pretest blood metabolites of NA pigs suggest that they were in a hypermetabolic state that predisposed them to becoming NA.

Anesthetics, Inhalation↗

PubMed2005

The effects of halothane sensitivity on carcass composition and meat quality in HAL-1843-normal pigs.

Objectives of this study were to determine the incidence of halothane sensitivity in pigs that are homozygous normal at the ryanodine receptor nucleotide 1843 (HAL-1843-normal) and the relationships between halothane sensitivity and carcass composition or meat quality. In Exp. 1, piglets (Lines A, B, C, and D; n = 168, 170, 168, and 169, respectively) were obtained from mating a HAL-1843-normal sire line to four HAL-1843-normal dam lines. In Exp. 2, piglets from Lines A and B (n = 87 and 90, respectively) were included with piglets (Lines E, F, G, and H; n = 94, 92, 89, and 89, respectively) obtained from mating four HAL-1843-normal sire lines to a single HAL-1843-normal dam line. Pigs were subjected to 3% halothane at approximately 9 wk of age. In Exp. 1, limb rigidity, blotching of the skin, and muscle tremors were visually assessed, and based on these criteria, halothane sensitivity (HS) was observed in 48% of the pigs. To better characterize this response, a scoring system was developed and used in Exp. 2. Using this system, 25, 42, and 33% of the pigs in E and 40, 33, and 27% of the pigs in Line G were categorized as HS-low (HS-L), HS-intermediate (HS-I), and HS-high (HS-H), respectively. In Lines F and H, 13 and 18% of the pigs were HS-I, and 0 and 2% were HS-H, respectively. No consistent effects due to HS were observed in carcass composition or meat quality; however, when a subset of pigs from Exp. 2 were subjected to more extensive handling and transportation before slaughter, ultimate pH was lower and drip loss was higher in LM from HS-H compared with HS-L pigs (P < 0.05; n = 71). These results demonstrate that some pigs are sensitive to halothane anesthesia even in the absence of the known HAL-1843 polymorphism. Additionally, halothane sensitivity may be associated with inferior pork quality under adverse antemortem conditions.

Anesthetics, Inhalation↗

PubMed2003

Effect of repeated administration of combination trenbolone acetate and estradiol implants on growth, carcass traits, and beef quality of long-fed Holstein steers.

Our objective was to determine the effect of repeated use of implants on feedlot performance and carcass characteristics of Holstein cattle. Holstein steers (n = 128) weighing an average of 211 kg were blocked by weight and randomly assigned to 16 pens. At the start of the trial (d 0), pens were assigned to one of four treatments: 1) nonimplanted control (C); 2) implant on d 0, 112, and 224 (T3); 3) implant on d 112 and 224 (T2); and 4) implant on d 224 (T1). Component TE-S implants (120 mg of trenbolone acetate and 24 mg of estradiol per implant) were used for all treatments during the 291-d feeding period. Over the course of the study, T2 and T3 cattle had greater ADG and final weights than C and T1 cattle (P < 0.05). Steers were harvested at a commercial abattoir on d 291. Hot carcass weights of T3 steers were greater than those of C and T1 steers (P < 0.05). Dressing percentage, adjusted 12th-rib fat, percentage of kidney, pelvic, and heart fat, yield grade, and longissimus color were not different among treatments (P > or = 0.26). Longissimus muscle areas (LMA) of T2 and T3 carcasses were larger than LMA of C (P < 0.01). No USDA Select carcasses were produced from C cattle, whereas the percentage of Select carcasses from implanted cattle ranged from 10 to 18%. Skeletal maturity advanced (P < 0.05) progressively with each additional implant. Steaks from T3 carcasses had a higher percentage of protein than controls (P < 0.05) and were less tender than all other treatments (P < 0.05). Repeated administration of combination trenbolone acetate and estradiol implants increased ADG and resulted in heavier carcasses with larger LMA. Administration of three successive implants decreased tenderness of Holstein beef, and resulted in more advanced skeletal maturity scores.

Anabolic Agents↗

PubMed2002

Satellite cell proliferation and differentiation during postnatal growth of porcine skeletal muscle.

Age-related changes in satellite cell proliferation and differentiation during rapid growth of porcine skeletal muscle were examined. Satellite cells were isolated from hindlimb muscles of pigs at 1, 7, 14, and 21 wk of age (4 animals/age group). Satellite cells were separated from cellular debris by using Percoll gradient centrifugation and were adsorbed to glass coverslips for fluorescent immunostaining. Positive staining for neural cell adhesion molecule (NCAM) distinguished satellite cells from nonmyogenic cells. The proportion of NCAM-positive cells (satellite cells) in isolates decreased from 1 to 7 wk of age. Greater than 77% of NCAM-positive cells were proliferating cell nuclear antigen positive at all ages studied. Myogenin-positive satellite cells decreased from 30% at 1 wk to 14% at 7 wk of age and remained at constant levels thereafter. These data indicate that a high percentage of satellite cells remain proliferative during rapid postnatal muscle growth. The reduced proportion of myogenin-positive cells during growth may reflect a decrease in the proportion of differentiating satellite cells or accelerated incorporation of myogenin-positive cells into myofibers.

Animals↗

PubMed2001

Biological and economic performance of early-weaned Angus steers.

Over 2 yr, 45 Angus-sired steer offspring of Angus and Angus crossbred females were used to determine the effects of early weaning on feedlot performance, carcass characteristics, and economic return to the cow-calf enterprise. Steers were assigned by birth date to one of two weaning treatments: 1) weaned at an average age of 100 d (early weaned) or 2) weaned at an average age of 200 d (normally weaned). Within 36 d of weaning, steers were given ad libitum access to a high-concentrate diet (90% dry, wholeshelled corn). Steers were harvested when 12th-rib fat thickness averaged 1.27 cm within treatment as estimated by ultrasound. Carcass measurements were taken 48 h postmortem and rib steak tenderness was determined at 14 d postmortem by Warner-Bratzler shear force. Early-weaned steers had greater ADG from time of early weaning to normal weaning than suckling normally weaned steers (1.27 vs. 0.86 kg/d, respectively; P < 0.001). However, early-weaned steers tended to have lower ADG for the entire finishing period than did normally weaned steers (1.33 vs. 1.39 kg/d, respectively; P = 0.08). Compared with normally weaned steers, early-weaned steers had lower daily DMI (7.40 vs. 5.95 kg/d, respectively; P < 0.001) and lower total DMI for the finishing period (1,618 vs 1,537 kg, respectively; P < 0.05). Early-weaned steers had greater gain:feed for the finishing period than normally weaned steers (0.223 vs 0.189, respectively; P < 0.001). Carcass weights were lighter for early-weaned steers than for normally weaned steers (277.9 vs. 311.2 kg, respectively; P < 0.001). There was no difference in yield grade (3.1 vs. 3.2; P < 0.10) between treatments. All carcasses graded Low-Choice or greater, and there was no difference in the percentage of carcasses grading Mid-Choice or greater (94.5 vs 83.9% for early- and normally-weaned, respectively; P > 0.10). Warner-Bratzler shear force values were similar between treatments. Early-weaned steers had a lower cost of gain than normally weaned steers ($ 0.82 vs. 0.91/kg, respectively; P < 0.001). However, due to lighter carcass weights, early-weaned steers generated less return to the cow-calf enterprise than normally weaned steers ($ 380.89 vs 480.08/steer; P < 0.001). The early weaning of steers at 100 d of age decreased total DMI, improved gain:feed, and lowered the cost of gain; however, return to the cow-calf enterprise was decreased due to lighter carcass weights.

Age Factors↗

PubMed1999

Immunoblot analysis of calpastatin degradation: evidence for cleavage by calpain in postmortem muscle.

A negative correlation exists between calpastatin activity and meat tenderness. Therefore, it is important to determine the mechanism of calpastatin inactivation in postmortem skeletal muscle. Western immunoblot analysis was performed to determine the protease(s) responsible for degradation of muscle calpastatin during postmortem storage. To accomplish this, purified calpastatin was digested with different proteases in vitro, and their pattern of calpastatin degradation was compared with that of calpastatin degradation in postmortem muscle. Polyclonal antibodies raised in mice against recombinant bovine skeletal muscle calpastatin were used to monitor calpastatin degradation. Lamb longissimus was stored at 4 degrees C and sampled at 0, 6, 12, 24, 72, 168, and 336 h postmortem. Postmortem storage produced a discrete pattern of calpastatin degradation products that included immunoreactive bands at approximately 100, 80, 65, 54, 32, and 29 kDa. Undegraded calpastatin (130 kDa) was barely detectable after 72 h of postmortem storage at 4 degrees C, and no immunoreactive calpastatin was observed by 336 h postmortem. For in vitro proteolysis, lamb longissimus calpastatin (0 h postmortem) was purified using Affi-Gel Blue chromatography. Calpastatin was digested with m-calpain, mu-calpain, cathepsin B, proteasome, trypsin, or chymotrypsin. Each of these enzymes degraded calpastatin. Immunoreactive fragments resulting from digestion of calpastatin with m- and mu-calpain were similar to each other and closely resembled those observed during postmortem aging of lamb longissimus at 4 degrees C. Digestion of calpastatin with mu-calpain reduced calpastatin activity. Degradation of calpastatin by other proteases resulted in unique patterns of immunoreactive fragments, distinct from that observed in longissimus. Thus, m- and(or) mu-calpain seem to be responsible for calpastatin degradation during postmortem storage of meat.

Animals↗

PubMed1998

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.

Animals↗

PubMed1996

Extrinsic regulation of domestic animal-derived satellite cells.

Satellite cells are the postnatal myogenic cells, as they provide myonuclei to support skeletal muscle hypertrophy and are principal cells responsible for myofiber repair and regeneration. Even though research with satellite cells from meat animals is new, considerable data exist to suggest that these cells are regulated through both intrinsic and extrinsic mechanisms. This review covers the present status of the extrinsic factors known or postulated to modulate meat animal satellite cell growth and development.

Animals↗

PubMed1996

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.

Animals↗

PubMed1996

Development of an enzyme-linked immunosorbent assay (ELISA) for quantification of skeletal muscle calpastatin.

An indirect antibody ELISA was developed for rapid and sensitive quantification of skeletal muscle calpastatin. Polyclonal antibodies were raised in rabbits against recombinant calpastatin, corresponding to domains 2, 3, and 4 of bovine skeletal muscle calpastatin. Western blot analysis revealed that these antibodies specifically recognize an immunoreactive calpastatin protein of approximately 130 kDa in prerigor skeletal muscle extracts. The intensity of the immunoreactive bands corresponds qualitatively with assayable calpastatin activity. For ELISA development, optimum dilutions of sample, primary anti-calpastatin antibody, and peroxidase-conjugated secondary antibody were determined by titration. A dilution optimum for coating of Immulon 4 (Dynatech) plates was observed when heated muscle extracts were diluted to 2 to 4 micrograms of protein/mL and incubated for 2 h at 37 degrees C. Optimum primary (30 micrograms IgG/mL) and secondary (Sigma A-6154; 1:1000 dilution) antibody incubations were for 1 h at 37 degrees C. Tetramethylbenzidine was used as substrate and A450 of the stopped reaction product was recorded in an automated plate reader. Calpastatin ELISA results were linearly related to calpastatin activity (calpain inhibitory activity) of heated longissimus muscle homogenates from prerigor lamb (r2 = .89; n = 40) and beef aged for 24 or 48 h (r2 = .90; n = 47). Intra-assay CV was < 5% (n = 8) and inter-assay CV was < 6% (n = 5). This assay offers advantages of speed, simplicity, and sensitivity over conventional methodology for calpastatin quantification.

Animals↗

PubMed1996

Meat toughening does not occur when rigor shortening is prevented.

The objective of this experiment was to test the hypothesis that meat toughening during the first 24 h postmortem results from sarcomere shortening during rigor mortis development. Eleven market-weight lambs were used to measure changes in shear force of clamped longissimus during rigor development. Within 15 min of exsanguination, while attached at both ends, each longissimus was separated from the vertebrae body and clamped between three sets of metal plates to prevent muscle shortening (six clamped sections per lamb). Five of the clamped sections were placed at -1.1 degrees C for 0, 3, 6, 12, or 24 h. After storage at their respective times at -1.1 degrees C, the samples were placed at -30 degrees C for 90 min and then at -5 degrees C for 8 d. The sixth section (168-h section) was stored at -1.1 degrees C for the first 24 h, at 4 degrees C for 144 h, and then treated the same as other sampling times. Sections were sampled for pH, sarcomere length, shear force, and Western blot analyses before and after storage at -5 degrees C. Shear force values were the same (P > .05) from 0 to 24 h (4.5 kg at 0 h to 4.9 kg at 24 h) then declined (P < .05) to 3.3 kg at 168 h postmortem. As evident by lack of statistical difference in the sarcomere lengths, we were successful in holding the muscle length constant. Western blot analyses of nebulin, vinculin, and troponin-T indicated that minimum degradation occurred through 12 h, was slightly increased by 24 h, and was relatively extensive by 168 h postmortem. Although limited proteolysis occurred during storage at -5 degrees C for 8 d, this by itself had no effect on shear force. Results indicate that shear force values do not increase during rigor development when muscle is prevented from shortening; thus, the toughening that occurs during the first 24 h of slaughter is most likely due to sarcomere shortening.

Animals↗

PubMed1995

A muscle hypertrophy condition in lamb (callipyge): characterization of effects on muscle growth and meat quality traits.

The present experiment was conducted to determine the effect of the callipyge phenotype on traits affecting muscle growth and meat tenderness. Dorset wethers (N = 40) that were either carriers or non-carriers were fed grain and slaughtered at 169 d of age. Callipyge phenotype did not affect (P > .05) slaughter weight, hot carcass weight, or weights of the heart, spleen, viscera, kidney-pelvic fat, head, and pelt; however, callipyge lambs had a higher dressing percentage and lighter lungs, liver, and kidneys (P < .01). Callipyge lambs had reduced fat thickness and marbling score and higher leg scores and longissimus area (34%). Adductor (30%), biceps femoris (42%), gluteus group (31%), longissimus (32%), psoas group (20%), quadriceps femoris (18%), semimembranosus (38%), and semitendinosus (26%) weights were higher in the callipyge phenotype (P < .01); however, phenotype did not affect (P > .05) weights of infraspinatus or supraspinatus. Longissimus pH and temperature declines were not affected (P > .05) by phenotype. Longissimus myofibril fragmentation index was lower at 1 (27%), 7 (35%), and 21 (37%) d postmortem and Warner-Bratzler shear force was higher at 1, 7, and 21 d postmortem in the callipyge phenotype (P < .01). Shear force values of callipyge lambs at 21 d postmortem tended to be greater (P = .12) than shear force values of non-carriers at 1 d postmortem . Activities of calpastatin (83%) and m-calpain (45%) were higher in the callipyge (P < .01); however mu-calpain activity was not affected (P > .05). Longissimus and semitendinosus RNA concentration, DNA content, RNA content, protein content, and the RNA:DNA ratio were higher (P < .05), but DNA concentration, protein concentration, and protein:DNA were not affected in the callipyge phenotype. The higher calpastatin activity associated with callipyge suggests that protein degradation may be reduced in the live animal. Additionally, the increased muscle DNA content associated with the callipyge phenotype suggests an increase in satellite cell proliferation, and results in an increased capacity of skeletal muscle to accumulate and maintain myofibrillar protein. These results suggests that both reduced rate of protein degradation and higher capacity for protein synthesis are consequences of the callipyge condition.

Actinin↗

PubMed1993

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.

Animals↗

PubMed1993

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.

Animals↗

PubMed1992

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.

Animals↗

PubMed1990

Satellite cells of growing turkeys: influence of donor age and sex on proliferation and differentiation in vitro.

Proliferation and differentiation (fusion) of myogenic satellite cells isolated from Nicholas tom and hen turkeys at 3, 9, and 15 weeks of age and the response of a satellite cell clone to serum from these birds were examined. Responsiveness of satellite cells to culture conditions was proportional to donor age, and sex of bird had no effect on satellite cell proliferation. Fusion percentages were similar in all cultures. When a turkey satellite cell clone was exposed to serum from tom and hen turkeys at each age, an age-related decline in proliferation occurred with serum from hen but not from tom turkeys. Minimal cell fusion occurred in turkey serum and neither proliferation nor fusion was correlated with serum IGF-I levels. Cells derived from pectoralis major and anterior latissimus dorsi muscles of 15-week-old tom turkeys possessed similar proliferation and fusion properties in vitro. It appears that satellite cells retain a high proliferative activity throughout growth in turkeys, and the mitogenic properties of turkey serum may be evaluated using turkey satellite cells in culture.

Aging↗

PubMed1990

Interaction of insulin-like growth factor I with turkey satellite cells and satellite cell-derived myotubes.

Satellite cells, isolated from the superficial pectoralis muscle of growing Nicholas tom turkeys, were cloned to obtain a pure population of myogenic cells. These cells proliferated rapidly and differentiated (fused) into myotubes typically containing 92-98% fused nuclei. Competitive binding assays were performed on near-confluent satellite cell or myotube cultures in 35 mm diameter wells by adding [125I]IGF-I along with increasing concentrations of unlabeled IGF-I, IGF-II, or insulin. Following incubation, the cultures were washed to remove the unbound hormones, solubilized with 0.5 N NaOH, and the radioactivity specifically bound was determined. Total and fused nuclei number as well as total protein were determined in parallel cultures. Our results indicate that turkey satellite cell and myotube cultures possess specific binding sites for IGF-I. Displacement of [125I]IGF-I was in the order of IGF-I greater than IGF-II greater than or equal to insulin. Although the [125I]IGF-I association constants were similar for turkey satellite cells and myotubes, a 2.8-fold decrease in the number of receptors per nuclei was observed as satellite cells differentiated into myotubes. The 50% inhibition constants for IGF-I, IGF-II, and insulin were 3.7 X 10(-9) M, 7.5 X 10(-8) M, and 8.7 X 10(-8) M for satellite cells and 3.1 X 10(-9) M, 7.5 X 10(-8) M, and 9.6 X 10(-8) M for myotubes, respectively. Receptor cross-linking analysis using disuccinimidyl suberate was performed on near-confluent satellite cell cultures incubated with [125I]IGF-I in the presence or absence of 1 X 10(-7) M IGF-I, IGF-II, or insulin. Receptor subunit species of Mr 130 kDa and 98 kDa were observed under reducing conditions (100 mM dithiothreitol) and at a Mr greater than 300 kDa (native receptor tetramer) under non-reduced conditions. Autoradiographic bands were displaced with IGF-I but not with equimolar levels of IGF-II or insulin. The results suggest that turkey satellite cells possess a type I IGF receptor.

Animals↗

PubMed1988

The turkey myogenic satellite cell: optimization of in vitro proliferation and differentiation.

Myogenic satellite cells were isolated from the pectoralis major muscle of young growing tom turkeys. These cells were capable of proliferating and forming large multinucleated myotubes in vitro. Of 36 media-sera combinations evaluated, McCoy's 5A medium containing 15% chicken serum (CS) promoted the greatest level of proliferation and subsequent myotube formation when cells were induced to differentiate (P less than 0.05). Myotube formation was maximized following exposure of cultures to Dulbecco's Modified Eagle's Medium (DMEM) containing 1% horse serum (HS; DMEM-1% HS) for 4 days. Satellite cells grown under these conditions generally resulted in cultures containing greater than 90% fused nuclei. Cells plated in the presence of DMEM-10% HS resulted in greater attachment and larger cultures (and consequently a greater fused nuclei number) when transferred to growth media than similarly grown cultures plated in McCoy's 5A medium-10% CS, regardless of substrata tested (P less than 0.05). The greatest proliferation and myotube formation was seen in cultures grown in gelatin-coated wells. Proliferation was maximized in McCoy's 5A medium containing 18% CS, although this was not significantly different than the proliferation with media containing 15% CS (P greater than 0.05). Our results (1) document that the postnatal myogenic satellite cell can be isolated from the turkey in sufficient quantities for biological studies and (2) identify culture conditions which optimize proliferation and differentiation of these cells in vitro.

Animals↗