287 IMPACT OF CULTURE ENVIRONMENT ON COLONY FORMATION OF CELLS ISOLATED FROM TESTES OF PREPUBERTAL AND ADULT BULLS

Establishment of culture systems with type A spermatogonial cells has focused primarily on the use of testicular cells from prepubertal animals. The objective of this study was to evaluate various culture systems on development of testicular cells derived from prepubertal and adult bulls. Testicular cells (were harvested from 3–4 mo-old prepubertal bulls (PB; Dairy; single fresh testis weight: 24.1 ± 0.1 g) and adult bulls (AB; Beef; 399.0 ± 0.5 g). After purification using a discontinuous Percoll density gradient, testicular cells (50 × 103 cells well–1; 24-well plate) were kept separate according to origin (prepubertal or adult) and were cultured in the presence (FL) or absence (NF) of a feeder monolayer (mitomycin C-treated male bovine fetal fibroblasts) with either embryonic-like stem cell media (ELSC; DMEM high glucose) or regular media (RCC; DMEM low glucose) supplemented with regular fetal bovine serum (FBS-S) or charcoal stripped (FBS-SF). Colony number, size and type (round, radial, and irregular) were examined on Days 4, 7 or 15 of culture. Data were analyzed using a randomized block design with factorial treatment arrangement. Depending on variable of interest, fixed treatment effects were feeder monolayer, fetal bovine serum type, media type, bull type, day of culture (4, 7, and 15 d) and interactions. Cell viability and percentage of germ and somatic cells at seeding were used as covariates. Plate (bull type × day) were random blocking factors. Cells from prepubertal bulls had greater viability than adult bulls (P < 0.0001) immediately after digestion (92.0 ± 0.1 and 84.9 ± 0.1%, respectively) and immediately after Percoll purification (78.8 ± 0.2 and 44.9 ± 0.2%, respectively). Following digestion and cell purification, percentage of cultured testicular cells staining positive for protein gene product 9.5 was 11.5 ± 0.17% for prepubertal bulls and 15.5 ± 0.19% for adult bulls (P < 0.0001). Colonies in culture presented similar morphological characteristics and timing of formation as those observed by Izadyar et al. (2003 Biol. Reprod. 68, 272–281) when culturing bovine type A spermatogonia from prepubertal bulls. Number of colonies increased from Day 4 to 15 of culture and was not affected by bull type. Overall, radial colonies were the most predominant type of colony in culture (P < 0.0001). The maximum number and size of colonies were obtained on a FL with RCC media containing FBS-S in prepubertal and adult bulls (P < 0.0001). Data illustrate that isolation and culture of testicular cells from mature bulls resulted in colony formation, similar to prepubertal animals. Although cell cultures were a mixed population, colonies with morphologies appearing similar to those previously reported in the literature suggest that a portion were likely stem cells in origin. Efforts are currently underway to use markers to further define colony populations. Nonetheless, these findings suggest that culture of testicular cells in the presence of a feeder layer with regular culture media containing FBS with steroids was beneficial in colony formation regardless of bull type.