R. A. Merkel

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.

The role of nitrite in preventing development of warmed-over flavour

Abstract Development of warmed-over flavour (WOF) was followed in samples of beef, pork and chicken with and without added nitrite. Samples were evaluated by the 2-thiobarbituric acid (TBA) test and by sensory panel scores both before and after cooking at 0 days and again after storage for 48 h at 4°C. Added nitrite inhibited WOF development in cooked meat, resulting in a two fold reduction in TBA values for beef and chicken and a five fold reduction in pork. Sensory panel scores confirmed the protective effect of added nitrite in meat from all three species. Total lipid levels were not significantly related to WOF, but there was evidence for involvement of phospholipids.

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.

Effects of salt and some antioxidants upon the TBA numbers of meat

Abstract The effects of salt alone (2%) or coated with α-tocopherol or with Tenox 4 (BHA-citric acid-propylene glycol) or in a mixture containing BHA and BHT with salt on the TBA numbers of raw and cooked beef were determined after holding for 0 or 2 days at 4°C. Salt accelerated lipid oxidation both during cooking and subsequent storage. The α-tocopherol-coated salt also increased lipid oxidation, but only during storage after cooking. Both Tenox 4-coated salt and the mixture of BHA and BHT with salt completely inhibited lipid oxidation in cooked meat, both during cooking and upon subsequent storage. Results suggest that selected antioxidants can be used to inhibit the development of warmed-over flavor (WOF) in cooked meats.

Influence of pH and temperature upon calcium accumulation and release by bovine sarcoplasmic reticulum

Abstract Sarcoplasmic reticulum (SR) was prepared from fresh beef sternomandibularis muscle and shown to be relatively free from contamination by lysosomes, sarcolemma and mitochondrial membranes. Ca2 + accumulation by SR from fresh and cold-shortened muscle was 51 and 39 nmoles/mg protein, respectively. The Ca2 + accumulating ability of fresh SR vesicles decreased with lowering of pH (7·3, 6·8, 6·2, 5·5 and 5·0) at all temperatures (0, 15 and 38°C). Lowering the temperature from 38 to 0°C at pH 6·6 resulted in the release of 48% of the total accumulated Ca2 +, whereas the corresponding value on lowering the temperature from 38 to 15°C at the same pH was only 12%. Thus, low temperatures accelerate the release of Ca2 + by SR. Although simultaneously lowering pH and temperature also increased Ca2 + release by SR, the amount of Ca2 + released was less than if pH and temperature were altered independently. The findings are discussed in the light of explaining cold shortening.

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.

RNA Transcription in Porcine Skeletal Muscle Nuclei During Postnatal Development

Abstract Postnatal developmental pretranslational regulation of skeletal muscle α-actin gene expression was investigated. Northern blot analysis of skeletal muscle α-actin and β-tubulin mRNA from 1- and 28-day-old pigs indicated that there are developmental increases in α-actin mRNA abundance (P < 0.03) and no significant changes in β-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°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 μg/ml α-amanitin to the transcription mixture. Transcription “run-on” assay results indicated that more (P < 0.02) skeletal muscle α-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 α-actin mRNA observed in the older animals was due, at least in part, to an increase in the transcriptional activity of the skeletal muscle α-actin gene.