This thesis describes studies on the spermatozoa of the ram carried out with a view to determining the effects of deep freezing
which lead to poor fertility following artificial insemination.
The electro-ejaculator was employed for semen collection over
20 months and 80 samples out of 114 collections were processed.
Motility of spermatozoa was assessed by scoring mass activity
in raw semen samples and by estimation of the percentage of motile
spermatozoa in raw and processed samples.
Percentages of live spermatozoa and their general morphology
were studied in eosin -nigrosin stained smears and acrosomal defects
in eosin fast green FCF stained smears. Spermatozoal morphology
was also studied with the electron microscope.
Generally the various parameters of the raw semen were within
or close to the standard ranges and they varied with season, best semen being collected in the autumn.
When semen was frozen by a standard technique there was a continuous reduction in the spermatozoal viability, especially
motility, associated with an increase in morphological deterioration
of the spermatozoa, especially of their acrosomes, following each stage.
At the same time variation in spermatozoal viability and morphology
existed between different samples.
Various modifications of the freezing technique were tried.
On inclusion of 2 -8% glycerol or dimethyl sulphoxide (DMSO), or their
combinations at different levels in an egg yolk and lactose diluent
the results respectively showed that 4% glycerol, 3% DMSO and 2%
glycerol with 1.5% DMSO were the optima.
The effects on spermatozoa of various equilibration times
(0.5 - 24.0 hours), dilution rates (1:1 - 1:10), thawing media (sodium
chloride or citrate with or without lactose in solution at 37 °C or frozen pellet at -196 °C) and thawing temperatures (0 °C - 100 °C) were
studied using both 4% glycerol or 3% DMSO as the cryoprotective in the
diluents. The results showed that equilibration of 3.0 hours with
glycerol, and of 1.5 hours with DMSO, and dilution rates of 1:4 and
thawing directly in a dry test tube at 37 °C - 100 °C, irrespective of
the cryoprotective, were the optima.
In addition, various methods of dilution (dropping or direct
at 4 °C or 20 °C or their combination) and different egg yolk levels
(25 or 50%) with or without sodium citrate (3%) were used. The
results indicated that equilibration at 20 °C, irrespective of method
of dilution led to a high death rate of ram spermatozoa but provided
surviving spermatozoa some resistance against cold shock during freezing. Direct addition of the diluent irrespective of the equilibration
temperature was satisfactory, but the addition of the diluent by
dropping for 0.5 hour at 20 °C followed by 1.0 hour equilibration at
4 °C was superior.
Increasing the egg yolk level from 25 to 50, with or without
sodium citrate in 4% glycerol containing diluent was harmful which
might be the result of binding up of glycerol.
Prompt dilution of the raw semen after collection and the
avoidance of temperature fluctuation between stages of freezing and
when sampling f,or thawing were tried and the results showed an enhanced
resistance to cold shock and an improvement in spermatozoal motility
when thawed after 24 hours storage at -196 °C. However, longer storage
X was still deleterious, and the percentage of motile spermatozoa fell
The post -thawing life span and morphological changes of the ram spermatozoa were evaluated following thawing at 0°C - 100°C and
incubation at 39°C for 0, 3.0 and 6.0 hours. The results showed
that the life span of the frozen -thawed ram spermatozoa was short
(around 3 hours) and their acrosomal defects increased progressively
as the incubation time increased.
The fertilizing efficiency of frozen semen stored for 42 - 46
days was tried on 28 ewes, but the results showed that the low
post -thawing motility and short survival time which pertained were
not adequate to produce pregnancy.