Abstract :
The maintenance of motility, viability and acrosomal integrity of ram spermatozoa for an
extended period of time after cryopreservation is important for achieving high pregnancy
rates, when using frozen–thawed semen. The most frequently used cryoprotectant is glycerol,
which has to be used at low concentrations (under 4%), due to its potential toxicity.
The primary objective of this study was to determine the combined effect of sub-optimal
glycerol inclusion and cholesterol-loaded cyclodextrin (CLC) in a Tris-based diluent on ram
spermatozoal longevity and acrosomal integrity during in vitro incubation, after cryopreservation.
Ram semen was treated with 0 or 2 mg CLC/100 × 106 cells in Tris-based diluents
containing either 6 or 3% glycerol and frozen in 0.5 ml straws. After thawing, the responses of
all sperm parameters recorded were not significantly different between sub-optimal glycerol
concentration (3%) and optimal glycerol concentration (6%) for untreated semen groups
(0 mg CLC/100 × 106). Similarly, no significant differences were recorded between glycerol
concentrations when semen samples were treated with 2 mg CLC/100 × 106 cells prior
to cryopreservation. However, sperm treated with CLC prior to cryopreservation in diluents
containing a sub-optimal concentration of glycerol (3%), recorded significantly higher
(P < 0.05) percentages of total motile sperm, progressive motility, viability and acrosomal
integrity, than for optimal glycerol concentrations (6%) for up to 4 h of in vitro incubation,
at 38.5 ◦C after cryopreservation and thawing. Results suggest that in ram spermatozoa, a
combination of 2 mg cholesterol-loaded cyclodextrins (CLC)/100 × 106 and a sub-optimal
concentration of glycerol (3%) in a Tris-based diluent helps to reduce the acrosomal damage
observed during cryopreservation, maintaining sperm viability and motility
