Semen cryopreservation has been of great benefit to livestock breeding, human infertility treatment and the conservation of endangered animals. However, the study of spermatozoa cryopreservation has been done empirically, using the complex extender containing egg yolk or skim milk. Besides being a potential source of infectious agent, these undefined components make it difficult to undertake proper quality control and to analyze the roles of a particular compound plays in sperm cryopreservation. Even so, only about 50% spermatozoa can survive during the freeze-thaw process, and the survivors' fertility is reduced. The rhesus monkey, one species of nonhuman primate, has been extensively used in biomedical research for long history. Cryopreservation of rhesus monkey spermatozoa would facilitate the breeding of this species and basic embryological research. Success with rhesus spermatozoa could also be useful in developing protocols for freezing sperm from other rare or endangered nonhuman primates. However, only a few reports have been published on cryopreservation of rhesus monkey spermatozoa, and effective methods for their quality evaluation are required. Thus, this investigation was devoted to establishing simple and effective methods for rhesus monkey spermatozoa cryopreservation and viability assessment. The main results were as follows. 1 An effective method for evaluating rhesus monkey sperm viability by flow cytometry using Hoechst 33342 and propidium iodide dual staining excited by a single UV laser was established and applied to the study of spermatozoa cryopreservation. 2 It was found that glycerol can be added or removed in a single or fractionated manner, but a single manner is easier and more practical Using the single manner, further experiments showed that extenders containing different sugars gave similar results, whereas TT extender with no sugar was more effective than sugar extenders in protecting sperm function after freezing and thawing. The reason was that the osmolalities of extenders were crucial to the cryoprotection of rhesus monkey spermatozoa. The osmolality of TT (138mOsm/kg) was favorable for the cryopreservation of rhesus monkey spermatozoa and the hyperosmotic stress of TEST accounts for the lower survival of rhesus monkey spermatozoa frozen in this medium. 3 In order to further optimize the freezing procedure, several key factors were investigated using TT as the basic extender. The results showed that the best sperm survival rate was obtained after freezing spermatozoa in 1 * TT containing 5% glycerol with 0.5h equilibration. Overall, post-thaw sperm motililty and viability were about 55% and 60%, respectively. The optimized method using TT yielded similar post-thaw results as did the conventional freezing method using the egg yolk based TTE extender, and the post-thaw sperm can be used successfully for in vitro fertilization. Furthermore, sperm ryopreservation efficacy could be further improved by diluting the liquefied semen directly with freezing medium at a rate of 1:2. (v/v). The establishment of the simple method using chemically defined medium should prove useful in future investigations to analyze the actual cryoprotecting potential of different reagents. The H342/PI dual staining technique, was simple and effective in assessing sperm viability and would be valuable for multiparameter flow cytometric analysis of sperm function.
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