Reproduction Abstracts

Porcine spermatogonia can develop to sperm in neonatal testicular tissue that were cryopreserved and grafted into nude mice. Live piglets were born from zygotes produced by intracytoplasmic sperm injection using these sperm (Kaneko et al. PLoS ONE, 2013). Utilization of fetal tissue will give us valuable chances for conservation of genetic resources and also for improvement of testicular xenografting. We aimed to examine whether porcine fetal testis can produce sperm after grafting into nude mice.

Testes obtained from 55- and 90-day-old crossbred fetuses (the date of artificial insemination was defined as day 0) were minced into fragments measuring 1.5×1.5×1.5 mm (F55 and F90 groups, respectively). Tissue fragments were incubated in vitrification solution (35% ethylene glycol, 5% polyvinylpyrrolidone and 0.3 M trehalose in a base solution) for 10 min at room temperature. They were then dropped with ~4 μl of vitrification solution into liquid nitrogen and were stored in liquid nitrogen. After storage, microdroplets containing tissue were transferred into warming solution (0.4 M trehalose in base solution) at 37 °C for 2 min then consecutively transferred for 2-min periods into 0.2, 0.1, and 0.05 M trehalose in base solution at room temperature. A total of 20 fragments were grafted subcutaneously into castrated nude mice.

Sperm recovery from the grafted tissue was confirmed in the F55 group at 180 days after grafting (1/5 mice). However, sperm recovery rate increased at 240 days (4/5 and 5/5 mice in the F55 and F90 groups, respectively) and at 300–420 days (7/10 and 13/14 mice, respectively).

These results demonstrate that fetal testis can produce sperm even after cryopreservation.