The Effect Of Cryopreservation On The Ultrastructure And Enzymes Activity Of The German Shepherd Dog Sperm

There have not been a matured set of semen cryopreservation technology that suitable for canine, so far. In this study we researched the normal ultrastructure of semen that taken from German shepherd dogs, and we also analyzed the changes in ultrastructure and acrosomal enzyme before semen cryopreservation and after semen cryopreservation with electron microscope. This study attempt to exploring an effective method of semen cryopreservation that suitable for German shepherd dogs, and to providing the theory basis for its future optimization as well as to promoting the development of semen cryopreservation. The result shows that,1. The German shepherd dogs fresh sperm ultrastructure observation. The thirty fresh semen from six German shepherd dogs was divided into six groups(Ⅰ～Ⅵ), and measured the quality of fresh semen respectively. Every fresh semen from six groups took500μl to make sample for scanning election microscope(SEM) and transmission election microscope(TEM). After observing and measuring the morphologic character of the samples from10-20fields of vision, we took pictures and record. We found that the sperm whole showed "tadpole-shape", with its head orbicularovate in the SEM. While in the TEM, its head showed "wedge-shape" and its cytomembrance was made up of plasmalemma, perforatorium ectoblast, perforatorium intima and karyotheca. The transection of the tail was made up of mitochondrial membrane,9binds of outerdensefiber,9pairs of axoneme and2centralmicrotubules from outside to inside. The semen head longth was5.40±0.12(μm), the head breath was3.26±0.02(μm), the neck longth was1.25±0.01(μm), the neck breath was0.55±0.02(μm),the middle tail longth was11.34±0.03(μm), the diameter was0.87±0.01(μm), the tail longth was55.70±0.22(μm) and the spiral number of the mitochondria was44±0.21(circle).2. The effect of cryopreservation on ultrastructure of German shepherd dogs sperm.Every the residual semen that from six groups took500μl to made into samples for SEM and TEM, after centrifugaling (1000r/min,10min)、diluting (1:1)、equilibrium (4℃,2h). The others was made into granular frozen semen, and made into samples for SEM and TEM after thawing (38℃,30s).After observing and measuring the morphologic character and size of the samples from10-20fields of vision, we took pictures and record. By contrasting the changes in ultrastructure of the sperm between before and after cryopreservation, we found that the changes of morphological changes in head, neck and tail of fresh semen and semen before cryopreservation were non significant(P>0.05). After cryopreservation, morphological changes in head, neck and tail were significanter than that in fresh sperm as well as sperm before cryopreservation. The sperm count that the perforatorium has a pronounced form change takes up54.21%, which higher than the sperm count of neck and tail,10.61%and28.83%, respectively.3. The effect of cryopreservation on acrosome enzymes activity of German shepherd dogs semen. After contrasting the changes in acrosome enzymes activity of the semen between before and after cryopreservation, we got that the acrosome enzymes activity were significant between the fresh semen and semen (P<0.01).4. The relativity between acrosome enzymes activity and the motility rate, acrosome integrity rate, and aberration rate. We analyzed the relativity between acrosome enzyme activity and motility rate, the acrosome integrity rate and aberration rate after semen cryopreservation, respectively; the result showed that the acrosome enzymes activity was positive correlation with the acrosome integrity rate and the motilityrate, especially the ACE activity was extremely positive correlation with the acrosome integrity rate with its correlation corefficient r=0.487(P<0.01). While the acrosome enzymes activity was negative correlation with aberration rate (P<0.05). The correlation corefficient of ACE activity and acrosome integrity rate was0.487, which higher than that of HYD(0.439and0.459) and ACP(0.440), while the correlation corefficient of HYD activity and the motilityrate was (0.404and0.423), which higher than that of ACE(0.377) and ACP(0.369).5. By the above results we infer that cryopreservation mainly cause damage to the membrance of the sperm head, especially to the perforatorium;which leads to the running off of the acrosome enzymes and the reduction of the enzyme activity, and then comes to a lower conception rate.