| In this study,semen samples collected from 4 angus bull were pooled and divided into 4 aliquots. Two of them (treatment 1 and treatment 2)were washed twice, with skin milk-egg-aminomethane-citric-acid and 0.9% Nacl respectively,the fluid was removed after centrifugation at 1000r/min 5 minutes each time. The third aliquot (treatment 3)was centrifugation at the same condition, but not being removed seminal plasma. The last aliquot (treatment 4)was treated as normal procedure, without centrifugation and removal of seminal plama. All four aliquots were treatmented wih skin milk-egg-aminomethane-citric-acid and were frozen in straws to -196 . The percentages of sperm motility, normal acrosome, abnormal spermaozoa, the concentration of GOT-realeased and the ability of penetration were investigated respectively. The result attained were as follow:1. In treatment 1 and treatment 2 ,the percentages of the frozen-thawed sperm of motility, normal acrosome, abnormal spermatozoa were consequently:0.493 0.043aad0.497 0.067, (78.3 2.7)%and(78.7 2.8)%, (6.35 0.72)%and (6.32 0.57) %. There were no-significant difference between them (P>0.05).In treatment 3 and treatment 4, the results were 0.312 0.053 and 0.313 0.062, (58.7 2.9) % and(58.9 2.9) %, (9.42 0.38) %and (9.39 0.82) %. There were no-significant difference too (P>0.05). But there were significant difference between the preceding two treatment and the latter ones (P<0.01). This result indicated that centrifugation had no-significant effect on the quality of post-thawed bovine spermatozoa,but removal of seminal plasma can improve it significantly.2. The content of GOT and LDH escaping from spermatozoa into the extra cellular plasma was used as an indicator of sperm cell membrane integrity. After removal of sperm plasma and dilution,the concentrations of GOT and LDH from spermatozoa (treatment 1 and 2) rose to (72.17 3.29) IU/ml and (71.98 4.05) IU/ml, (284.1 31.34) lU/ml and (281.9 45.1) lU/ml .The data (treatment 3 and 4) rose to (52.45 5.18) lU/mland (51.83 7.01) IU/ml, (208.7 23.6) lU/ml and (206.9 + 39.2) lU/ml. There were significant difference between the preceding two treatments and the latter ones(P<0.05). After post- thawing, the four aliquots had no significiant difference in the content of GOT and LDH. These resaults indicate that the integrity of sperm cell membrane was changed by removal of seminal plasma. At the same time , the removal of seminal plasma could protect the sperm cell membrane when being frozen.3. TEM was used to investigate the ultrastructure change of bovine spermatozoa before and after being frozen. It was observed that centrifugation gave rise to the lightly swollen of membrane of spermatozoa. Removal of plasma had no effect on the ultrastructure before being frozen. Centrifugation also did not work to the ultrastructure ,but the acrosome of spermatozoa after being frozen was damaged badly. The two treatment of removal of sperm plasma have remarkable decreasing to the ultrastructural damage. This result showed that removal of sperm plasma could improve the capability of spermatozoa in resisting freeze.4. Detection of the fertilization ability of bovine frozen-thawed sperm by penetrating the zona-free ova of hamster. The treatment 1 and 2 had signficantly higher penetration rate (84.5 7.8)%and(85.1 6.2)%than the treatment 3 and 4 owned (70.4 12.3)%and(71.1 9.7)%.The two treatment being removed sperm plasma had higher averages of spermatozoa penetrating ova(2.93 0.35)and( 2.91 0.57)than the other two ones (1.58 0.43)and(1.70 0.81). The results proved that the removal of seminal plasma from bovine semen was beneficial in spermatozon cryopreservation and can improve the fertilization ability of Angus bovine spermatozoa. |
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