Effect of processing and freezing procedures on the acrosome morphology of buck spermatozoa
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Date
1998
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Department of Animal Reproduction, College of Veterinary and Animal Sciences, Mannuthy
Abstract
Six pooled semen samples (two ejaculates) of good quality from five
Malabari crossbred bucks were processed and frozen in two different
protocols to evaluate the effect of processing and freezing procedures on
the acrosome morphology of buck spermatozoa.
In protocol I, the samples were diluted 10 fold in Tris buffer before
centrifuging twice and the final pellet was re-suspended in the non
glycerolated fraction of Tris yolk diluent. The sample was glycerolated
(six per cent), equilibrated (four hours), frozen (eight minutes), and
thawed (250 C for 30 seconds). In protocol 11, centrifugation was done only
once, after 15 fold dilution in Tris buffer. The re suspended pellet was
glycerolated (seven per cent), equilibrated (three hours), frozen (10
minutes) and thawed (60° C for 10 seconds). The semen characters such
as motility, live sperm, sperm abnormalities and acrosome abnormalities
were evaluated at the end of washing and initial extension (stage I),
cooling to 5° C (stage II), glycerolisation and equilibration (stage Ill) and
freezing and thawing (stage IV). The results were compiled to evaluate
the effect of different processing and freezing procedures on the semen
characters in general and acrosome morphology in particular.
The semen sample used for split sample dilution had a mean volume of
1.3282± 0.067 ml, creamy in colour, DDDD density, ++++ mass activity, pH
of 7.275 2± 0.040 and a concentration of 2972 2± 293 millions per ml. No
significant difference in the above semen characters were found between
bucks.
The initial sperm motility of 82.000 2± 0.606 was found to drop significantly
during processing and freezing and the final post thaw motility obtained
was 44.000 2± 0.790 in protocol I. Similarly in protocol II the initial motility
dropped from 81.375 2± 1.089 to 44.750 2± 1.075 at the end of stage IV.
Even though there was significant drop in motility between stages in both
the protocols, there was no significant difference in the corresponding
stages of the two protocols. It could be inferred that good post thaw
motility was obtained in both the protocols. The fact that a single washing
and centrifugation was only adopted in protocol II makes it a more
acceptable procedure for buck semen freezing.
The mean live sperm percentage of fresh semen was evaluated using
both NE and NEG staining technique. The percentage of live sperms of
90.050 2± 0.801 was found to decrease to 54.250 2± 0.593 after freezing
and thawing in protocol by NE staining. Similarly in protocol 11, the mean
percentage of live sperms was found to reduce to 53.125 2± 0.793 with the
same staining. Even though there was significant difference in the live
sperm percentage between stages within protocol I and II no significant
difference in the live sperm percentage between the corresponding stages
of protocol I and I I . With NEG staining the initial live sperm percentage of
80.850 ± 1.494 was found to drop to 54.875 ± 0.677 in protocol I as
against 53.400 ± 0.730 in protocol II. While there was significant
difference in the live sperm percentage between stages within protocol I
and II there was no variation between corresponding stages of the two
protocols. A significantly lower percentage of live sperms was recorded
with NEG staining when compared with NE staining probably on account
of the fact that the differentiation of live and dead sperm was difficult in
the former staining method as live sperms were stained light blue instead
of colourless.
The mean percentage of abnormal sperms of 3.050 ± 0.245 in fresh
semen did not register any significant increase during processing.
However, there was significant increase in the percentage of sperm
abnormalities during freezing and thawing with the final abnormality
percentage of 7.125± 0.706 in protocol I and 6.300± 0.36 in protocol II.
The initial acrosomal abnormality of 8.825 in the fresh semen steadily
rose to 23.375 in protocol I as against 19.825 in protocol II at the end of
stage IV. There was no significant difference in the percentage of various
acrosomal abnormalities between corresponding stages of the two
protocols. However, there was significant increase in the acrosomal
abnormalities during glycerolisation, equilibration, freezing and thawing
under both the protocols. It was concluded that the processing and
freezing under two different protocols did not significantly alter the post
thaw motility, percentage abnormal and dead sperms and acrosomal
abnormalities. A good post thaw motility and low acrosomal abnormality
was obtained with a single washing of buck semen with 15 fold Tris buffer
which was comparable with double washing with 10 fold Tris buffer.
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MVSc
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171368