| The object of this study was to analyse the semen quality of Guangxi Debao pony (stallion) in different seasons, so as to guide the production of semen cryopreservation and breeding of Debao pony. Moreover, the influencing factors of Debao pony semen cryopreservation were also studied, including the basic formula of the semen freezing extenders, cryoprotectant, nutrient, vitamin etc, so as to obtain an optimized formula of the Debao pony semen freezing extender, and get higher quality of frozen semen. In addition, the applying effect of the frozen semen on the production practice was tested by artificial insemination (AI). The research results had been summarized as follows:1. The semen quality of Debao pony appeared the best quality in the spring season, which sperms had a higher motility, density, and a lower deformity rate. The semen quality in the winter and autumn season was the second one, and the summer season was the worst one. Although the ejaculate volume of Debao pony was largest in the summer season, but the sperm motility and the density were lowest, and the abnormal rate was highest, the results showed that the semen quality of Debao pony presents seasonal difference. So, the suitable season of semen collection for cryopreservation was in the spring season.2. The effects of five different formulas of semen freezing extenders (Formula numbers were1,2,3,4,5) on semen cryopreservation of Debao pony were compared. The results showed that the post-thaw sperm motility of the formula2was significantly higher than the other four formulas (P<0.05). The movement rate (VAP, VCL) of the post-thaw sperm was also significantly higher than the formula1,3,4(P<0.05). Therefore, the effect of the formula2on semen cryopreservation of Debao pony was preferable. Thus, the formula2was used as the basic formula for the following experiments.3. On the basis of formula2, the effects of the different cryoprotectants (glycerol and DMF) on semen cryopreservation of Debao pony were evaluated. The results indicated that the effect of using5%DMF for pony semen cryopreservation treatment was better than adding6%glycerol group (P<0.05).4. To explore the effects of the soy lecithin on semen cryopreservation of Debao pony, different concentrations of the soy lecithin (1.5%,3.0%,4.5%,6.0%) were respectively used to substitute the yolk in the semen freezing extender. The results showed that there was no significant difference in the post-thaw sperm motility between the low concentration of soy lecithin group (1.5%,3.0%) and the control group (5%yolk group)(P>0.05). The post-thaw sperm motility of the high concentration soy lecithin group was slightly lower than the control group (P<0.05). The movement rate (VAP, VSL, VCL) and the acrosome integrity of post-thaw sperm in the low concentration group (1.5%,3.0%) were higher than the control group (P<0.05). The sperm abnormal rate in the different concentration of soy lecithin group was higher than the control group (P<0.05). Moreover, the post-thaw sperm survival time in vitro of the every soy lecithin concentration group was lower than the control group (P<0.05). So, the results showed that soy lecithin can not instead of the yolk in the semen freezing extender of Debao pony.5. The effects of adding vitamin E and vitamin C into the semen freezing extender on the semen cryopreservation were examined, and found that the motility, acrosome integrity, survival time in vitro of post-thaw sperm were significantly higher than the control group as adding600μg/ml vitamin E into the freezing extender (P<0.05), especially the survival time in vitro was much longer, though the movement rate and the abnormal rate of the post-thaw sperm had no significant differences with the control group (P>0.05). When vitamin C was added to the frozen diluted solution, there were not significantly different in the motility, movement rate, abnormal rate and the acrosome integrity of post-thaw sperm comparing with the control group (P>0.05), although the survival time in vitro of the post-thaw sperm was significantly higher than the control group (P<0.05), but was lower than the vitamin E adding group.6. To study the effect of artificial insemination of frozen semen. The frozen semen of0.5ml straws were produced with the optimized formula derived from above experiments, and22oestrus cycles of16mares were inseminated by AI with this frozen stallion semen. Seven mares were confirmed to be pregnant by the rectal palpation, the pregnant rate of the total oestrus cycle was31.8%. It showed that Debao pony could be pregnant after AI with this frozen semen, despite the pregnant rate was lower. Study shall be necessary for improving the pregnant rate of Debao pony after AI with frozen semen.These results indicated that:(1) The semen quality of Debao pony was better in the spring season, and it was suitable to collected semen for cryopreservation.(2)5%DMF could be substituted glycerol as the cryoprotectant for Debao pony semen cryopreservation, moreover, adding600μg/ml vitamin E into the freezing extender could significantly improve the quality of Debao pony frozen semen.(3) The optimized formula of Debao pony semen freezing extender (100ml) was included with lactose (7.2g), glucose (3.6g), citric acid sodium (0.4g), yolk (5.0ml), DMF (5.0ml), vitamin E (0.06g) and penicillin/streptomycin (hundred thousand units), and that Debao pony could be pregnant after AI with frozen semen yielded by this optimized formula. |
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