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Cryopreservation of Spermatozoa in Tree Shrews and Rhesus Monkeys

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Tutor: YangShiHua
School: Kunming University of Science and Technology
Course: Biochemical Engineering
Keywords: sperm,rhesus monkey,tree shrew,cryopreservation,ultrastructure
CLC: S865.1
Type: Master's thesis
Year:  2011
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Sperm is an essential genetic resource for mammalian. Sperm cryopreservation play an important role in fast propagation of experimental animals, livestock breeding, biodiversity conservation as well as human infertility and sterile. Tree shrews and rhesus monkeys are widely used around the world in research because of their genetic evolution and physiological similarities to humans. However, reproductive biology and cryobiology of most species, and the mechanisms of sperm cryo-injury, and the cryo-protective mechanisms of some compounds remain poorly understood, which cause a dramatical decrease in survival and fertility of post-thaw sperm. Therefore, it is essential to investigate the characteristics of sperm cryopreservation and factors affecting cryo-survival of mammalian sperm to improve cryopreservative efficiency.The tree shrews and rhesus monkeys were used as animal models for collecting sperm in this experiment. Sperm motility and acrosome integrity were examined to evaluate frozen-thawed sperm function. In addition, artificial fertilization (AI) or in-vitro fertilization (IVF) was used to evaluate fertilizability of frozen-thawed sperm. The present study demonstrated that:(1) Effects of egg-yolk concentrations, freezing rates and holding times in liquid nitrogen vapor on epididymal sperm cryopreservation of tree shrew; (2) Effects of four penetrating cryoprotectants on cryopreservation of tree shrew epididymal sperm; (3) Sperm ultrastructure of tree shrew was observed by scanning electron microscope(SEM) and transmission electron microscopy(TEM); (4) Optimize ethylene glycol concentrations, freezing rates and holding times in liquid nitrogen vapor on cryopreservation of Rhesus monkey sperm; (5) Effect of seminal plasma on rhesus sperm cryopreservation.The results showed that:(1) Tree shrews epididymal sperm were successfully cryopreserved in TTE containing 20% egg yolk at-172℃/min for 10 min and retained their viability during cryostorage and the subsequent fertilizing ability by AI; (2) 3% Dimethylsulphoxide (DMSO) is the most suitable for tree shrew epididymal sperm cryopreservation of the four penetrating cryoprotectants (Glycerol, Ethylene glycol, DMSO, Propylene glycol), which significantly improved the viability of frozen-thawed sperm. Moreover, the frozen-thawed sperm have the ability to fertilize oocytes in vivo using intrauterine artificially insemination; (3) Tree shrew sperm had a round or oval head in shape of approximately 6.65x5.82μm, and midpiece, principal piece, tail, and total sperm lengths were 13.39,52.35,65.74, and 73.05μm respectively. The tail modpiece mitochondria consisting of approximately 48 gyres has a 9+9+2 pattern of axonemal. It has similar ultrastructure with other mammalians except for mitochondria number and the size of sperm; (4) Optimal EG concentration, freezing rate and freezing time in liquid nitrogen vapor for the cryopreservation of rhesus sperm were 4%,-183℃/min, and 10min, respectively. Most importantly, sperm cryopreserved with 4% EG were able to fertilize rhesus macaque oocytes in vitro, and the fertilization rate and embryo development rate were similar to that with 5% Gly. These findings provided an alternative penetrating CPA for genetics preservation purposes in this important species; (5) Seminal plasma is beneficial to sperm to undergo cold shock or cryopreservation in individual monkeys. The cryosurvival of sperm from rhesus monkey with poor sperm freezability could be improved by presenting seminal plasma from males with good sperm cryosurvival.The present study established an efficient method for collecting, diluting, and preserving tree shrew sperm, and using tree shrews fresh and frozen-thawed sperm to fertilize oocytes in vivo by intrauterine artificially insemination. We investigated the characteristics of sperm cryopreservation and factors affecting cryo-survival in tree shrews sperm to improve its propagation under breadboard condition, which offered basic knowledge to understand embryo biology and establish transgenic tree shrews. Meanwhile, the study on sperm cryopreservation in rhesus monkey provided a useful method for genetic preservation with an improvement frozen-thawed sperm cryosurvival and fertilizability in this important species. In addition, the results will also be vital for researchers to understand and investigate the mechanisms of sperm cryo-injury and cryoprotection of some compounds and also contribute to the areas of biodiversity preservation, germplasm resource cryopreservation, and cryobiological development.
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