A procedure is described that allows cryopreservation and efficient po
st-thaw recovery of either a single or a small group of human spermato
zoa, This is achieved by injecting them into cell-free human, mouse or
hamster zonae pellucidae before the addition of cryoprotectant. The m
ethod involves a combination of physical micromanipulation procedures
and glycerol-mediated cryoprotection. Zonae were tracked by positionin
g them in straws between two small air bubbles prior to freezing, Sper
matozoa from poor specimens were cryopreserved and their fertilizing a
bility after thawing was compared with that of fresh spermatozoa from
fertile men, Human eggs used for fertilization testing were either 1 d
ay old or in-vitro matured, Only 2% of the frozen zonae were lost and
>75% of spermatozoa cryopreserved in this manner were recovered and pr
epared for intracytoplasmic sperm injection, The feasibility of cryopr
eserving a single spermatozoon was assessed, Fifteen motile spermatozo
a were frozen in 15 zonae, of which 14 were recorered after thawing. T
en were injected into spare eggs, of which eight became fertilized, Sp
ermatozoa recovered mechanically from human zonae fertilized the same
proportion of oocytes as fresh fertile control spermatozoa, The recove
ry and fertilization rates with spermatozoa frozen in animal zonae mer
e 87 and 78% respectively, The fertilization rate was marginally highe
r (P < 0.05) than that for spermatozoa frozen in human zonae, perhaps
because the latter may have acrosome reacted more frequently, The zona
pellucida appears to be an ideally suited sterile vehicle for storage
of single spermatozoa.