Evaluation On Sperm Viability, Membrane And Acrosome Integrity In Blue Fox Semen During Low-tempreture Storage

Artificial insemination is an important reproductive biotechnology for fox breeding now. It is valued by fox breeder more and more during genetic improvement in native blue fox with Finnish giant blue fox. With the biotechnology development, the storage of semen is more important in vitro. Artificial insemination with fresh semen is used now in practical production, and can maintain good fertility. But fresh semen diluted in vitro remains viable for a limited period of time, the resoures of excellent male blue foxes are not used enough, which lead to the excellent semen was wasted largely. The cryopreservation of blue fox semen is still difficult for large-scale use due to the low sperm viability following thawing and low fecundity rates after artificial insemination. The storage of blue fox in low temperature can reduce the damages of cryopreservation, but remain viable for a longer time than in normal temperature, and can be used in artificial insemination. However, the fertility of semen is gradually lost during the low temperature storage. Therefore, the storage in low temperature and the proper characterization of blue fox during the low temperature storage is of utmost importance.In this study, freshly ejaculated blue fox semen was diluted with IVT( Ⅰ), Glycine(Ⅱ), Egg yolk-milk(Ⅲ) or Glucose-egg yolk(Ⅳ) extender, and storage at 0~5℃ for up to 5 days. Sperm quality in blue fox during the low temperature storage was evaluated by examing viability with eosin-nigrosine staining, membrane integrity by Hypoosmotic Swelling Test, and acrosome integrity using Coomassie blue staining, but was compared with DongLin II that is a kind of normal temperature extender. Results show the ratio of viability, membrane integrity, acrosome integrity in low temperature storage is higher than in normal temperature. Both eosin-nigrosine staining and Hypoosmotic Swelling Test were sensitive for evaluating sperm quality. The result assessed with eosin-nigrosine staining was lower than that with Hypoosmotic Swelling Test. The viability and the ratio of membrane integrity determined by eosin-nigrosine staining and Hypoosmotic Swelling Test were lower than the percentage of acrosome integrity with Coomassie blue staining. The results from eosin-nigrosine staining, Hypoosmotic Swelling Test and Coomassie blue staining were highly correlated (r≥0.9981). The percentages of viable sperm, membrane integrity, acrosome integrity during the low temperature storage were slowly reduced up to 2 days, after that were reduced quickly; which changed slowly before 12 hours during the normal temperature storage, after that changed quickly. The percentages of viable sperm, membrane integrity, acrosome integrity during the normal temperature storage were better than that during in low temperature storage up to 8 hours. The percentages of viable sperm were 48.81 ±3.21% for Ⅰ, 63.62±2.47% for Ⅱ, 42.10 ± 2.68% for Ⅲ, 55.07±2.79% for Ⅳ and 14.60±1.85% for Dong Lin Ⅱ extenders on day 3 of storage respectively. The percentages of viable sperm were under 10% in others except Ⅱ extender up to 5 days. The percentages of viable sperm, membrane integrity.acrosome integrity during the low temperature storage extender I > UK IV on day 2, II extender on day 4 and during the normal temperature storage extender on day 1 were over 60% in semen, respectively. The semen still can be used in artificial insemination. In conclusion, the rank order of four low-temperature extenders for maintaining sperm viability, membrane integrity and acrosome integrity was as follows: II>IV> I >III. They were all better than normal-temperature extender.Additionally, the viability, survival time and survival index of spermatozoa from nine male blue foxes were compared in low and normal temperature storage in this study. The survival time of blue fox spermatozoa were prolonged markedly in vitro during the low temperature storage (0~5"C), and had better viability after improving the temperature. The longest survival time and highest survival index of spermatozoa were 50 hours, 27.4; and average survival time and survival index were 24.22 hours, 12.7 during the normal temperature storage, respectively. The longest survival time and highest survival index of spermatozoa werel08 hours, 48.4; and average survival time and survival index were 37 hours, 17.89 during the low temperature storage, respectively. The survival time and survival index of blue fox semen during the normal temperature storage and those during the low temperature storage were correlated in line. The storage result of blue fox semen during the normal or low temperature were different among different foxes and in different period. The quality of semen has widely declined in final reproductive period. The semen were not suitable to store in the low temperature.