FREEZABILITY OF BUCK SPERMATOZOA TREATED WITH CHELATING AGENT FOR FIBRONECTIN TYPE II PROTEINS
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Date
2011
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COLLEGE OF VETERINARY AND ANIMAL SCIENCES-MANNUTHY,THRISSUR
Abstract
A study to evaluate the effect of Fn2 type protein chelating agent
(choline chloride) or immediate extension and washing on the freezability of
buck spermatozoa was carried out at Artificial Insemination Centre, under the
department of Animal Reproduction, Gynaecology and Obstetrics, College of
Veterinary and Animal Sciences, Mannuthy, Thrissur using 18 normal
ejaculates from an adult healthy Malabari crossbred buck. The total seminal
plasma protein content was estimated by Lowry’s method. The ejaculates
were grouped into three with group I consisting of ejaculates collected directly
into Tris extender alone, group II consisting of ejaculates collected into Tris
extender containing choline chloride and group III serving as the control. The
processed semen was packed in 0.25 ml French straws after extending them in
Tris yolk glycerol extender and cryopreservation was carried out.
The volume of buck semen samples collected under group III
(control) were 0.57 ± 0.09 ml and average sperm concentration was 3971 ±
99.42 million per ml. The gross semen characteristics except volume (0.6 ±
0.09 ml and 0.32 ± 0.03 ml respectively) and sperm concentration (3683 ±
115.49 and 3905 ± 147.50 million per ml) could not be evaluated directly
from semen samples of group I and group II as these were collected directly
into a collection vial containing Tris extender and choline chloride solution
respectively. Mean progressive motile spermatozoa of the three groups were
83.83 ± 1.25, 82.67 ± 1.26 and 84.67 ± 0.84 per cent respectively, while the
mean live sperm percentage were observed to be 94.83 ± 0.83, 94.17 ± 1.08,
95.33 ± 0.61 respectively in the three groups. Average per cent of abnormal
spermatozoa of the three groups were 3.00 ± 0.63, 3.00 ± 0.68 and 2.50 ±
0.56 respectively. Mean per cent of acrosomal integrity of spermatozoa were
90.50 ± 0.99, 90.17 ± 1.17 and 89.67 ± 0.95 respectively in three groups.
After equilibration, the percentage progressive sperm motile
spermatozoa in group I, II and III (control) were 76.17 ± 1.08, 77.17 ± 1.08
and 73.67 ± 1.15 respectively. The viable spermatozoa percentage of three
groups observed at prefreeze stage were 82.67 ± 1.36, 83.33 ± 0.76 and 79.33
±0.99 respectively which did not show any significant difference between the
groups. The intact acrosome percentage at prefreeze levels were 83.67 ± 1.12
in group I, 82.00 ± 0.97 in group II and 82.67 ± 0.95 in group III. The sperm
abnormality of the three groups were 3.33 ± 0.56, 3.33 ± 0.42 and 3.50 ± 0.67
respectively. The mean of membrane integrity by hypo osmotic swelling test
of group I, group II and group III were 67.67 ± 2.04, 65.17 ± 2.02, 61.17 ±
1.66 respectively. Spermatozoa of group I had significantly higher HOS
response than spermatozoa of group III (control).
After freezing and thawing, the mean percentage of progressively
motile spermatozoa were found to be 40.83 ± 0.91 in group I, 44.67 ± 1.31 in
group II and 33.83 ± 1.30 in group III. The post-thaw live spermatozoa were
49.17 ± 0.91, 52.17 ± 1.30 and 44.17 ± 1.40 respectively. The values
obtained in both the treatment groups differed significantly from the values
observed in the control group. The values of intact acrosome in percentage
were 65.83 ± 1.35 in group I, 71.00 ± 1.00 in group II and 63.33 ± 1.05 in
group III. The spermatozoa of choline chloride treated group had significantly
higher intact acrosome than spermatozoa of both group I and control group.
The percentage of sperm abnormality obtained after thawing were 11.33 ±
0.56 in group I, 10.67 ± 0.49 in group II and 11.17 ± 0.60 group III
respectively. The mean of membrane integrity by hypo osmotic swelling test
of group I, group II and group III were 43.83 ± 2.50, 48.17 ± 2.30, 37.17 ±
1.89 respectively. Significant difference (p<0.05) was obtained between teh
HOS response of spermatozoa of choline chloride treated group and the
control group.
It was observed that both immediate extension and washing of
Malabari buck spermatozoa or treatment with choline chloride at the time of
semen collection resulted in improved sperm post thaw characters. Of the two
techniques, treating spermatozoa with choline chloride at the time of
collection resulted in better post thaw motility, viability acrosome integrity
and HOS response of frozen buck semen.
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