Abstract :
This study was conducted to elucidate the effect of increasing the osmolality of a basic
Tris, extender supplemented with sucrose, trehalose or raffinose on post-thawing ram
semen quality (sperm motility, viability, acrosome integrity, total sperm abnormalities
and membrane integrity). After primary evaluation of the collected ejaculates, only semen
samples with more than 70% motile sperm, and a sperm concentration of higher than
3×109 sperm/ml were used for cryopreservation. The semen samples were pooled and
diluted (1:4) with a Tris–citric acid–fructose–yolk extender, supplemented with different
concentrations (50, 70 or 100mM) of sucrose, trehalose or raffinose. As control, semen was
diluted and frozen in the base diluent, without additional sugars. Pooled semen samples
were aspirated into 0.25 ml straws, cooled to 5 ◦C within 90 min and frozen by exposure to
liquid nitrogen vapor (4–5cm above the liquid nitrogen surface) for 10 min – before plunging
into liquid nitrogen, for storage. After 24 h, straws were thawed in a water bath (37 ◦C)
for 30 s. The frozen-thawed sperm characteristics were improved significantly (P < 0.05) by
increasing the level of the sugars. Optimal results being obtained with 70 and 100mM trehalose
or raffinose. All extenders containing supplemental sugars were superior in terms
of sperm quality to the control (P < 0.01) group. The highest sperm motility (60.6±1.9%),
viability (60.6±2.5%) and membrane integrity (58.2±2.1%) were recorded using 100mM
trehalose and the lowest with 50mM sucrose (48.6±1.9%, 51.4±2.5% and 47.9±2.1%,
respectively). All sugar concentrations decreased the percentage of acrosomal and total
sperm abnormalities (P < 0.05). The extenders containing 100mMtrehalose or raffinose significantly
(P < 0.05) decreased the occurrence of sperm abnormalities, compared to the other
treatments. The fertility rates obtained after cervical insemination of the frozen-thawed
sperm were 46.8%, 44.1% and 16.7% for 100mM trehalose, 100mM raffinose and the control
with supplementation of the diluents, respectively. The study showed that ram sperm
can tolerate hyperosmotic diluents, and that a range of sugar concentrations (50–100mM)
may successfully be incorporated in the ram semen cryopreservation diluents, although
further research is warranted.
