• 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.

• The Korean Journal of Nuclear Medicine Technology
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• v.16 no.2
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• pp.162-166
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• 2012

Purpose : Vitamin $B_{12}$ and Folate are for anemia work-up which is well known for its sensitivity of light; the screening manual also specifies to be careful with light conditions. According to this, our laboratory minimized the exposure of light when inspecting the Vitamin $B_{12}$ and Folate, but the exposure cannot be wholly blocked due to other various factors such as when conducting specimen segregation. Thus, this inspection is to identify to what extent light can influence and whether the exclusion of light is mandatory during the Vitamin $B_{12}$/Folate test. Materials and Methods : We have conducted two experiments of identifying the extent of light's influence when conducting the Vitamin $B_{12}$/Folate test and also when specimens are under preservation. These experiments were progressed with various concentrations of patients' specimens which were requested to our hospital in March 2012. The first experiment is to verify the results on Vitamin $B_{12}$/Folate dependent on light exposure during the experiment. In the process, we have compared the results of light exposure/exclusion during the incubation process after the reagent division. The second experiment is about the impact of light exposure on the results on Vitamin $B_{12}$/Folate during the preservation. For 1, 2, 7 days the light on the specimen were wholly blocked and were preserved under $-15^{\circ}C$ temperature refrigeration. Then, we compared the results of light-excluded specimen and the exposed one. Results : When conducting first experiment, there were no noticeable changes in the Standard and specimen's cpm, but for Vitamin $B_{12}$, the average result of specimen exposed to light increased 7.8% compare to that of excluded one's. Furthermore, in the significant level 0.05, the significance probability or the p-value was 0.251 which means it has no impact. For Folate, the result being exposed to light decreased 5.4%, the significance probability was 0.033 which means it has little impact. For the second preservation, the result was dependent on the light exposure. The first day of preservation of Vitamin $B_{12}$, the clinical material exposed to light was 11.6%, second day clinical material exposed to light was 10.8%, seventh day clinical material exposed to light increased 3.8%, the significance probability of the $1^{st}$, $2^{nd}$, $7^{th}$ day is 0.372, 0.033, 0.144 respectively, and which indicates that the $1^{st}$ and $7^{th}$ day seems to have no impact. For Folate's case, the clinical material exposed to light has increased 1.4% but hardly had impact, $2^{nd}$ day clinical material being exposed to light was 6.1%, $7^{Th}$ day clinical material being exposed to light decreased 5.2%. The significance probability of Folate on the $1^{st}$, $2^{nd}$, $7^{th}$ day is 0.378, 0.037, 0.217 respectively, and the $1^{st}$ day and the $7^{th}$ day seems to have no impact. Conclusion : After scrutinizing the impact of light exposure/exclusion, Vitamin $B_{12}$ has no impact, while Folate seems to have no noticeable influence but light exclusion is recommended due to its significance probability of 0.033 when conducting experiment. During the preservation, the $2^{nd}$ day result depend on the light exclusion seems to have impact or influence. However, to consider the complication of the experimental process, the experiment including technical errors is predictable. Hence, it is likely to have no impact of light. Nevertheless, it is recommendable to exclude the light during the long preservation as per the significance probability (p-value) of $1^{st}$ and $7^{th}$ day has been diminished.

• Journal of Korean Society of Forest Science
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• v.21 no.1
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• pp.1-34
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• 1974

The author intended to investigate external and internal changes in the cone structure, changes in water content, sugar, fat and protein during the period of seed maturation which bears a proper germinability. The experimental results can be summarized as in the following. 1. Male flowers 1) Pollen-mother cells occur as a mass from late in April to early in May, and form pollen tetrads through meiosis early and middle of May. Pollen with simple nucleus reach maturity late in May. 2) Stamen number of a male flower is almost same as the scale number of cone and is 69-102 stamens. One stamen includes 5800-7300 pollen. 3) The shape is round and elliptical, both of a pollen has air-sac with $80-91{\mu}$ in length, and has cuticlar exine and cellulose intine. 4) Pollen germinate in 68 hours at $25^{\circ}C$ with distilled water of pH 6.0, 2% sugar and 0.8% agar. 2. Female flowers 1) Ovuliferous scales grow rapidly in late April, and differentiation of ovules begins early in May. Embryo-sac-mother cells produce pollen tetrads through meiosis in the middle of May, and flower in late May. 2) The pollinated female flowers show repeated divisions of embryo-sac nucleus, and a great number of free nuclei form a mass for overwintering. Morphogenesis of isolation in the mass structure takes place from the middle of March, and that forms albuminous bodies of aivealus in early May. 3. Formation of pollinators and embryos. 1) Archegonia produce archegonial initial cells in the middle and late April, and pollinators are produced in the late April and late in early May. 2) After pollination, Oespore nuclei are seen to divide in the late May forming a layer of suspensor from the diaphragm in early June and in the middle of June. Thus this happens to show 4 pro-embryos. The organ of embryos begins to differentiate 1 pro-embryo and reachs perfect maturation in late August. 4. The growth of cones 1) In the year of flowering, strobiles grow during the period from the middle of June to the middle of July, and do not grow after the middle of August. Strobiles grow 1.6 times more in length 3.3 times short in diameter and about 22 times more weight than those of female flower in the year of flowering. 2) The cones at the adult stage grow 7 times longer in diameter, 12-15 times shorter diameter than those of strobiles after flowering. 3) Cone has 96-133 scales with the ratio of scale to be 69-80% and the length of cone is 11-13cm. Diameter is 5-8cm with 160-190g weight, and the seed number of it is 90-150 having empty seed ratio of 8-15%. 5. Formation of seed-coats 1) The layers of outer seed-coat become most for the width of $703{\mu}$ in the middle of July. At the adult stage of seed, it becomes $550-580{\mu}$ in size by decreasing moisture content. Then a horny and the cortical tissue of outer coats become differentiated. 2) The outer seed-coat of mature seeds forms epidermal cells of 3-4 layers and the stone cells of 16-21 layers. The interior part of it becomes parenchyma layer of 1 or 2 rows. 3) Inner seed-coat is formed 2 months earlier than the outer seed-coat in the middle of May, having the most width of inner seed-coat $667{\mu}$. At the adult stage it loses to $80-90{\mu}$. 6. Change in moisture content After pollination moisture content becomes gradually increased at the top in the early June and becomes markedly decreased in the middle of August. At the adult stage it shows 43~48% in cone, 23~25% in the outer seed-coat, 32~37% in the inner seed-coat, 23~26% in the inner seed-coat and endosperm and embryo, 21~24% in the embryo and endosperm, 36~40% in the embryos. 7. The content compositions of seed 1) Fat contents become gradually increased after the early May, at the adult stage it occupies 65~85% more fat than walnut and palm. Embryo includes 78.8% fat, and 57.0% fat in endosperm. 2) Sugar content after pollination becomes greatly increased as in the case of reducing sugar, while non-reducing sugar becomes increased in the early June. 3) Crude protein content becomes gradually increased after the early May, and at the adult stage it becomes 48.8%. Endosperm is made up with more protein than embryo. 8. The test of germination The collected optimum period of Pinus koraiensis seeds at an adequate maturity was collected in the early September, and used for the germination test of reduction-method and embryo culture. Seeds were taken at the interval of 7 days from the middle of July to the middle of September for the germination test at germination apparatus.