• Current Research on Agriculture and Life Sciences
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• v.7
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• pp.83-97
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• 1989

This study was carried out to obtain basic information necessary for aggregation and in-vitro culture of mouse embryos by treating phytohemagglutinin-p (PHA-P). The 4-, 8-cell and morula embryos were obtained from female mice of albino BALE/C, CBA and C57BL strains, those were injected 5 i.u pregenant mare serum gonadotrophin and 5 i.u human chorionic gonadotrophin to superovulation. The zona pellucidia was removed by placing the embryos in Acidic Tyrode solution containing 1.0% protease or/and 5 ug/ml PHA-P. The pairs of zona free embryos were subjected to aggregation by glassneedle in BMOC-3 containing 5 ug/ml PHA-P. The aggregation embryos were cultured in Brinster's mouse ova culture-3(BMOC-3) medium under the gas phase of 5% $CO_2$ in air $37^{\circ}C$ for 13 to 50 hours. The results obtained in this study are summarised as follows : 1. When 4-, 8-cell and morula embryos were zona-freed in acidic Tyrode solution containing 1.0% protease or/and 5 ug/ml PHA-P, and cultured in vitro to blastocysts, the 4- and 8-cell embryos showed slightly less development rates than the morula one did, and solution of 5 ug/ml PHA-P brought some higher development rate than negative control. 2. As 2, 5 or 10 ug/ml PHA-P was added to the solution to aggregate 4-, 8-cell or morula embryos, 2 ug/ml solution represented slightly lower aggregation rate than the higher levels solutions, and 4- and 8-cell embryos showed higher rates than morula one did (P<.05). 3. In respect to the development rates of aggregated embryos to morula no significant difference was found among PHA-P levels and between 4-and 8-cell embryos. With respect to those of aggregated embryos to blastocysts the different levels of PHA-P showed similar results, however, the 4- and 8-cell embryos represented higher rates than the morula one did (P<.05). 4. The mean time necessary for development of aggregated 4-, 8-cell and morula embryos to blastocysts were 38.5-40, 26-27 and 19-20hrs. Respectively in solution for aggregation. 5. The aggregation rates of embryos were 34-94%, when treated protease or/and PHA-P. Supplementation of 5 ug/ml PHA-P to the solution for aggregation showed a trend demonstrating higher aggregation rate compared to negative control, although no significance was found. However, 4- and 8-cell embryos represented significantly higher aggregation rates than the morula one did (P<.05). 6. The development rates of 4- and 8-cell embryos to morula were 52.7-84.7 and 73.8-87.2%, respectively, showing no significant difference between two cell stages. However, the aggregation rates of embryos treated with solution containing PHA-P were higher than negative control (P<.05). 7. The development rates of 4- and 8-cell and morula embryos to blastocysts were 41.7-77.7 78.7-83.0 and 0-19.2%, respectively. The rates of 4-cell embryos treated with PHA-P were significant higher than the negative control (P<.05). The 8-cell and morula embryos also showed more rates when treated PHA-P.

• Korean Journal of Fisheries and Aquatic Sciences
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• v.25 no.2
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• pp.79-92
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• 1992

This study was carried out to develop new techniques useful for cryopreservation, thawing and artificial insemination, and ultrastructural changes of cryopreserved spermatozoa in rainbow trout(Oncorhynchus mykiss) . Two extenders, such as Tyrode solution and Whittingham's $T_6$ solution, were used to preserve rainbow trout sperm in refrigerator $(-20,\;-40\;and\;-70^{\circ}C)$ or liquid nitrogen $%(-196^{\circ})$. Hand-stripped semen was diluted to 1:16 with two extenders, an then the semen were frozen after mixing semen and each extender containing 1M or 1.5M DMSO solution to 1:1. After 60 days cryopreserved semen was thawed in a $13^{\circ}$ water bath, and subsequently centrifugated. After centrifugation at 1,000 rpm for 5 min thawed semen was washed with extenders, and then fertilized with fresh eggs. The results obtained in these experiments were summarized as follows: After cryopreservation, over 75% of spermatozoa were appeared motile and the survival rate was high. Following cryopreservation by the addition of cryoprotectant such as DMSO, methanol and glycerol, the fertilization rate of the thawed spermatozoa appeared over $99\%$ compared with the control having $99\%$ of fertilization rate. There was no difference between the control and experimental groups such as $(-20^{\circ}C\;-40^{\circ}C\;and\;-70^{\circ}C)$ and $-196^{\circ}$ in fertilization rate. Following cryopreservation at $-196^{\circ}$ by the addition of 1M DMSO of cryoprotectant, each fertilization rate following 24 hours and hatching rate following 24 days showed $96\%$ and $8\%$ by the addition of BSA, but showed $98\%\;and\;10%$ by no addition of BSA. Following 2 months of cryopreservation by the addition of 1M DMSO of cryoprotectant, there were $10%$ of hatching rate at $-196^{\circ}\;and\;10\%\;and\;35\%,\;respectively,\;at\;-40^{\circ}C\;and\;-70^{\circ}C$. Following 2 months of cryopreservation by the addition of 1M methanol of cryoprotectant, there were $22\%$ of fertilization rate at $-20^{\circ}C,\;and\;28\%,\;at\;-70^{\circ}C$ Following 2 months of cryopreservation by the addition of 1M glycerol of cryoprotectant, there were $22\%$ of fertilization rate at $-20^{\circ}C$, and $33\%,\;at\;-70^{\circ}C$. pollowing 2 months of cryopreservation by the addition of 1.5M DMSO of cryoprotectant, there were $27\%$ of fertilization rate at $-20^{\circ}C,\;an\;36\%\;and \;35\%,\;respectively,\;at\;-40^{\circ}C\;and\;-70^{\circ}C$. Following 2 months of cryopreservation by the addition of 1.5M glycerol of cryoprotectant, there were $34\% \;of\;fertilization\;rate\;at\;-20^{\circ}C, \;and\;31\%\;and\;31\%,\;respectively,\;at \;-40^{\circ}C\;and\;-70^{\circ}$. Following 2 months of cryopreservation by the addition of 1.5M methanol of cryoprotectant, there were $28\%$ of fertilization rate at $-20^{\circ}C,\;and\;29\%\;and\;28\%,\;respectively,\;at\;-40^{\circ}C\;and\;-70^{\circ}C.$ From 10 days and 15 days following fertilization at $13^{\circ}C\;and\;10^{\circ}C$, respectively, the mortality rate of fertilized ova was markedly increased. The middle piece of spermatozoa had two set of central doublets, nine set of outer coarse fibres, and mitochondrial sheath. Spermatozoa went through morphological changes during storage, e.g. winding of flagella, detachment of the nuclear envelope and the plasma membrane from the nucleus of the sperm head. There were $1\%$ abnormal spermatozoa in fresh sperm and about $15\%$ during storage.