The functional status of boar spermatozoa undergoing capacitation in v
itro was investigated. Two fluorescent stains were used: chlortetracyc
line (CTC) and a FITC-conjugated lectin (FITC-PSA). The first has been
used for the direct identification of the capacitated boar spermatozo
a, while the second, based on the identification of capacitated sperma
tozoa by their ability to undergo zona-induced acrosome reaction (AR),
was used to confirm and validate the CTC assay in this species. Sperm
atozoa obtained from 5 different boars was washed and incubated under
capacitating conditions. Aliquots of spermatozoa were collected at 0,
90 and 180 min of incubation and then stained with CTC or FITC-PSA. Af
ter CTC staining, 3 different fluorescent patterns were observed: Patt
ern A with the fluorescence uniformly distributed on the sperm head, P
attern B with the fluorescence concentrated in the post-acrosomial reg
ion, and Pattern C with the fluorescence concentrated in the acrosomia
l region. The percentage of spermatozoa displaying fluorescent Pattern
A decreased throughout the incubation while that of spermatozoa with
Pattern C showed a concomitant progressive increase. Pattern B fluores
cence remained unchanged throughout the maturation period. Exposure to
zonae pellucidae (ZP) brought back the levels of Pattern C fluorescen
ce to basal values. Since only the capacitated spermatozoa are believe
d to react to ZP, this observation together with the rising incidence
of Pattern C throughout maturation suggests that fluorescence in the a
crosomial region identifies capacitated spermatozoa. The analysis of a
crosome integrity carried out with FITC-PSA showed that the proportion
of zona-induced AR was nearly the same as that of spermatozoa display
ing Pattern C, thus confirming that CTC staining is suitable for the d
etection of boar sperm capacitation. In the second part of this study,
CTC was used to investigate the effects of sperm origin and storage o
n the capacitation process. Our finding demonstrates that capacitation
kinetics show wide variations In sperm samples derived from different
boars; moreover, capacitation is also affected by sperm storage. Whil
e fresh semen showed a progressive increase in capacitated spermatozoa
, ranging from low levels at the beginning of the culture to 46 % at t
he end of incubation, the refrigerated semen had a relatively high per
centage of capacitated spermatozoa at the beginning of culture, but th
is proportion increased only slightly during the following 90 to 180 m
in of treatment. These data indicate that CTC can be used to identify
capacitated boar spermatozoa, and, because of its rapid and easy execu
tion, it can be used routinely to identify the optimal capacitation ti
me for different sperm samples.