RESEARCH ARTICLE Slow Freezing, but Not Vitrification Supports Complete Spermatogenesis in Cryopreserved, Neonatal Sheep Testicular Xenografts

The ability to spur growth of early stage gametic cells recovered from neonates could lead to significant advances in rescuing the genomes of rare genotypes or endangered species that die unexpectedly. The purpose of this study was to determine, for the first time, the abili-ty of two substantially different cryopreservation approaches, slow freezing versus vitrifica-tion, to preserve testicular tissue of the neonatal sheep and subsequently allow initiation of spermatogenesis post-xenografting. Testis tissue from four lambs (3-5 wk old) was pro-cessed and then untreated or subjected to slow freezing or vitrification. Tissue pieces (fresh, n = 214; slow freezing, then thawing, n = 196; vitrification, then warming, n = 139) were placed subcutaneously under the dorsal skin of SCID mice and then grafts recovered and evaluated 17 wk later. Grafts from fresh and slow frozen tissue contained the most ad-vanced stages of spermatogenesis, including normal tubule architecture with elongating spermatids in ~1 % (fresh) and ~10 % (slow frozen) of tubules. Fewer than 2 % of seminifer-ous tubules advanced to the primary spermatocyte stage in xenografts derived from vitrified tissue. Results demonstrate that slow freezing of neonatal lamb testes was far superior t