107 INITIAL RESULTS FROM MALE GERM CELL TRANSFER BETWEEN CATTLE BREEDS

Abstract

Male germline cell transfer has produced offspring in mice (Brinster & Zimmerman 1994 PNAS 91, 11 298–11 302). Recently the first livestock animal, a goat, was produced (Honaramooz et al. 2003 Mol. Reprod. Dev. 64 422–64 428.), and early results in cattle are promising (Izadyar et al. 2003 Reproduction 126, 765–774; Oatley et al. 2002 J. Anim. Sci. 80, 1925–1931). We have assessed the outcome of male germ cell transfer between breeds of cattle and the efficacy of two vital dyes as markers of donor cells following transfer. Testis cells from three Bos taurus (Angus) bull calves were used as donor cells to transfer into six Bos indicus cross (predominantly Brahman bloodline) bull calves. Each of the calves was prepubertal and aged between 5 and 7 months. The calves were castrated; then a single-cell suspension of testis cells was prepared enzymatically using collagenase, DNAase, and trypsin. Prior to transfer into the recipient calves, the testis cell suspensions were dyed with one of two long-term vital dyes (PKH26 or CFDA). Approximately 300 million cells were injected into the rete of each testis under ultrasonographic guidance. In four of the six recipients, CFDA was injected into one testis and PKH26 into the other. These four recipients were castrated at 2, 4, 6, and 8 weeks after transfer. The other two recipients received either CFDA or PKH26 into both testes and were castrated at 8 weeks after transfer. Following castration, PKH positive donor cells were found in freshly isolated tubules of each of the five recipients that received PKH-dyed cells, while no CFDA-positive donor cells were conclusively identified in any of the recipients. In the freshly isolated tubules, clumps of PKH-positive donor cells were observed, which indicated either cell division or substantial local colonization of certain areas of the tubules. Frozen sections were used to further localize the PKH positive donor cells. Positive cells were located on the seminiferous tubule basement membrane, which indicates these cells had successfully migrated from the tubule lumen and were likely to be spermatogonia. There was variation in the amount of fluorescence for individual cells, which indicated either cell division or variable uptake of the stain during the staining procedure. We were disappointed to find no conclusive evidence of CFDA stained cells as we encountered high background fluorescence from the majority of testis cells. Although this fluorescence was quenched within 10 s, we were unable to find positive cells with any certainty. We have concluded that PKH26 was more suitable for labeling donor testis cells and that donor cells can be identified for at least 2 months following transfer. Each of the recipients that received PKH26 stained cells retained these cells in the tubule epithelium, which suggests that transfer between different animals, and indeed between breeds, can be achieved. Further studies will aim to demonstrate that donor cells are able to undergo spermatogenesis in the recipient animals.