021. Current progress into testis cell transfer between cattle breeds

Male germline cell transfer has produced offspring in mice (Brinster and Zimmermann 1994). Recently the first livestock animal, a goat, was produced (Honaramooz et al. 2003), while early results in cattle are promising (Oatley et al. 2002; Izadyar et al. 2003). There is an opportunity to develop this technology for the beef industry by transferring male germ line stem cells between breeds to improve the genetics of extensive Australian beef herds. This project is a part of the CSIRO National Research Flagship program that combines expertise and facilities in divisions with complementary expertise at Monash University and the University of Sydney. The environmental constraints of Northern Australia dictate that Brahman type animals show far better survival than Bos taurus cattle, although the carcass value of Brahmans is lower than Bos taurus animals. Artificial insemination is impractical in Northern Australia and thus we aim to develop testis cell transfer technique in cattle to permit Brahman bulls to deliver semen from elite Bos taurus or composite bulls, thereby significantly increasing the growth rate, yield and meat quality of the northern beef herd. Experiments using cattle were performed to determine the applicability of techniques used in the mouse. Initial proof of concept has been achieved that germ cell transfer can result in the donor cells successfully colonizing areas of recipient testis. The viability of isolated testis cells following short term (24 h) culture has been demonstrated through transfer into recipient calves. We have completed >50 male germ cell transfers into recipient calves, using ultrasonographic guided injection into the rete testis. Success of this procedure has been demonstrated by persistence of PKH26 dyed donor cells in the seminiferous tubules of a majority of recipients >2 months after transfer. These recipient male calves have not been depleted of their endogenous spermatogonial populations and we thus expect the efficiency of the procedure to increase as depletion procedures (ongoing) are established. Concurrent with these developments has been research into large scale culture of male germ line stem cells to provide large numbers of stem cells for transplant. Culture of testis cell suspensions has demonstrated survival of enriched testis cells under varying media and culture conditions. Initial passaging of testis cell colonies has revealed mixed cell populations (immunohistochemistry positive for spermatogonia and somatic cells). Further studies will aim to demonstrate that these cultured donor cells are able to undergo spermatogenesis in the recipient animals.