In vitro measurement of post-natal changes in proliferating satellite cell frequency during rat muscle growth

Satellite cells, resident myogenic stem cells found in postnatal skeletal muscle, are most abundant during early postnatal development and sharply decline in frequency thereafter to adult levels in mice and rats. Therefore, postnatal changes in satellite cell mitotic activities are important aspects for further understanding a muscle growth strategy. In large meat-production animals, however, the traditional in vivo proliferation assay may be less realistic because it requires intra-peritoneal (ip) injection of huge dosage of mutagenic nucleosides, (3)H-labeled thymidine or bromodeoxyuridine (BrdU), at each age-time of sacrifice. We report in the present pilot study using rats that in vivo proliferation activity of satellite cells can be evaluated by an in vitro BrdU-incorporation assay in early cultures. Briefly, satellite cells were prepared from upper hind-limb and back muscles and maintained for 24 h with imposing by BrdU addition for the last 2 h, followed by the regular immunocytochemistry for determining BrdU-incorporated cell percentage. This in vitro assay demonstrated a rapid decrease in proliferating satellite cell frequency to the adult level during about 3-month period after birth, and yielded a high correlation to the measurements by the in vivo BrdU ip-injection method during the postnatal period examined from day-2 to month-11. The in vitro proliferation assay may be further adaptable for large domestic animals by the combination with a muscle biopsy technique that enables age-interval sampling from the same growing animals.