• Clinical and Experimental Reproductive Medicine
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• v.21 no.1
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• pp.69-76
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• 1994

The survival and pregnancy rates were compared between non-frozen embryos and cryopreserved embryos at either pronucleate or 2-4 cell stages using the freezing and thawing techniques being identical in both groups were compared with fresh embryos. 496 embryos were frozen with 1, 2-propanediol and sucrose and 117 2-4 cell stages embryos had been thawed and 79.6 and 66.0% of them respectively were survival. Clinical pregnancy rate was 19.2% for embryos frozen at the pronucleate stage and 19.0% for embryos frozen at the 2-4 cell stages while the pregnancy rate of non-frozen embryos was 21.3%. There were no significant difference in the survival and pregnancy rates of embryos frozen at pronucleate and 2-4 cell stages. The current cumulative pregnancy rate per retrieval in all cycles with frozen zygotes is 35.4 %, consid~ erably higher than observed in single transfers of embryos without cryopreservation(21.3%); predicted pregnancy rate after transfer of all frozen embryos is 43.3 %. It is concluded that firstly, the survival and pregnancy rate of cryopreserved embryos at pronucleate or 2-4 cell stages are very similar to those from their fresh embryos and non-frozen embryos and secondly, cryopreservation substantially enhances pregnancy attainment from in vitro fertilization.

• Journal of Marine Life Science
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• v.4 no.2
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• pp.63-69
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• 2019

This study aims to investigate how ovulation induction of red spotted grouper, Epinephelus akaara varies when treated with different hormonal substances such as ovaprim, pimozide, LHRHa, and HCG. As for LHRHa, we injected it at four different concentrations: 50 ㎍kg^-1, 100 ㎍kg^-1, 150 ㎍kg^-1, and 200 ㎍kg^-1. All hormonal substances including LHRHa were injected into the back muscles. Buoyant, embryonic survival, fertilization, and hatching rate were the highest in LHRHa and LHRHa + pimozide group. When LHRHa was injected at different concentrations, buoyancy and fertilization rate were the highest in 100 ㎍kg^-1 group. Embryonic survival and hatching rate were the highest in 150 ㎍kg^-1. Based on these experimental results, we concluded that LHRHa at the concentration of 100 ㎍kg^-1 is the most efficient for the ovulation induction in this fish species.

• The Korean Journal of Malacology
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• v.26 no.3
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• pp.201-209
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• 2010

The investigated of survival rate, sex ratio, condition index, gonad development frequency, gonad index, fertilization rate, hatching rate and time required development of D shape larvae of the ark shell, Scapharca subcrenata. Survival rate was lower in heating group compared with control group (P < 0.05). Sex ratio (female:male) observed 1:0.97 in control group, 1:0.62 in heating group. Condition index was significantly higher in heating group than control group (P < 0.05) and higher increased more than water temperature $17^{\circ}C$ of heating group. Gonad development observed slow in control group but more than ripe stage 80% in heating group (21-28 day, $20^{\circ}C$). Gonad index was higher in heating group compared with control group (P < 0.05). Fertilization rate and hatching rate of the ark shell was observed $82.17{\pm}3.55%$ and $63.57{\pm}2.36%$, respectively. The time required development of D shape larvae of the ark shell was observed 15 hour after fertilization. The suggested that results of the ark shell by heating were effective sex maturation and normal egg development.

• Journal of Aquaculture
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• v.8 no.3
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• pp.241-249
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• 1995

Sperm from mud loach (Misgurnus mizolepis) were electroporated in the presence of plasmid DNA, pRSV/luc or pMT/hGH over a range of field strength of 0-1,625 V/cm with capacitance from 0 to 1,000 ${\mu}F$, and the effects of electroporation on fertilization, hatching, early survival, and efficiency of gene transfer were investigated. Average fertilization rate, hatching rate and early survival rate up to yolk sac absorption of all experimental groups were not significuntly different (P>0.05). The proportion of fish carrying pRSV/luc based on the polymerase chain reaction (PCR) analysis was ranged from 0 to $20\%$, however, the values of gene transfer efficiency from the different eledctroporation conditions were not significantly different. PCR analysis of pMT/hGH transferred groups revealed that screening of pMT/hGH transferred fish by PCR was difficult because of significant nonspecific amplifications resulted from the homologous sequences in the genome of mud loach.

• Journal of Fisheries and Marine Sciences Education
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• v.29 no.2
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• pp.409-414
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• 2017

Effects of human chorionic gonadotropin (HCG) on ovulation and spawning of the grass puffer, Takifugu niphobles, were investigated. Matured females spawned successfully by all doses of HCG. Spawning usually occurred 24 hours after hormone injection. Body weight of adult, gonadsomatic index (GSI), pseudo-GSI, body weight of spawned egg, success rate of spawn, fertilization rate, hatching rate, and survival rate of juvenile were correlated with increasing HCG doses. However, abnormal rate of juvenile was not correlated with increasing HCG doses. These results should be useful for developing aquaculture program of grass puffer.

• Asian-Australasian Journal of Animal Sciences
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• v.15 no.4
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• pp.485-493
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• 2002

Different factors may affect the sensitivity of porcine oocytes during cryopreservation. The effect of two methods (cooling and vitrification), four cryoprotectants [glycerol (GLY), 1, 2-propanediol (PROH), dimethyl sulfoxide (DMSO) or ethylene glycol (EG)] and two vitrification media (1 M sucrose (SUC)+8 M EG; 8 M EG) on the developmental capacity of porcine oocytes at the germinal vesicle (GV) stage or after IVM at the metaphase II (M II) stage were examined. Survival was assessed by FDA staining, maturation and cleavage following IVF and IVC. A toxicity test for different cryoprotectants (GLY, PROH, DMSO, EG) was conducted at room temperature before cooling. GV and M II-oocytes were equilibrated stepwise in 1.5 M cryoprotectant and diluted out in sucrose. The survival rate of GV-oocytes in the GLY group was significantly lower (82%, p<0.01) than that of the other group (92 to 95%). The EG group achieved a significantly higher maturation rate (84%, p<0.05) but a lower cleavage rate (34%, p<0.01) than the DMSO group and the controls. For M II-oocytes, the survival rates for all groups were 95 to 99% and the cleavage rate of the GLY group was lower than the PROH-group (21 vs 43%, p<0.01). After cooling to $10^{\circ}C$, the survival rates of GV-oocytes in the cryoprotectant groups were 34 to 51%, however, the maturation rates of these oocytes were low (1%) and none developed after IVF. For M II-oocytes, the EG group showed a significantly higher survival rate than those of the other cryoprotectant groups (40% vs 23-26%, p<0.05) and the cleavage rates of PROH, DMSO and EG group reached only 1 to 2%. For a toxicity test of different vitrification media, GV and M II-oocytes were equilibrated stepwise in 100% 8 M EG (group 1) and 1 M SUC + 8 M EG (group 2) or equilibrated in sucrose and then in 8 M EG (SUC+8 M EG, group 3). For GV-oocytes, the survival, maturation and cleavage rates of Group 1 were significantly lower than those in group 2, 3 and control group (p<0.05). For M II-oocytes, there were no differences in survival, maturation and cleavage rates between groups. After vitrification, the survival rates of GV and M II-oocytes in group 2 and 3 were similarly low (4-9%) and none of them matured nor cleaved after in vitro maturation, fertilization and culture. In conclusion, porcine GV and M II-oocytes do not seem to be damaged by a variety of cryoprotectants tested, but will succumb to a temperature decrease to $10^{\circ}C$ or to the process of vitrification, regardless of the cryoprotectant used.

• Clinical and Experimental Reproductive Medicine
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• v.26 no.2
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• pp.171-178
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• 1999

The follicular fluid (FF) of ovary contains various biological active products which affected on the growth of follicles and the fertilization of oocyte in physiological reproductive process of mammals. This study was designed to determine the effects of human FF on fertilization of oocyte and embryonal development in vitro culture. The FF was prepared as clear without blood contamination by needle aspiration from mature follicles of human at the time of oocytes retrieval for in vitro fertilization (IVF). As the medium for culture in vitro of embryonal cells, human tubal fluid (HTF) supplemented with follicular fluids at concentrations of 10%, 40% and pure FF were used. These effects were compared to control group of cultured embryos in HTF supplemented with 0.4% BSA (bovine serum albumin). For IVF, 64 eggs in control group, 67 eggs in 10% FF, 57 eggs in 40% FF and 64 eggs in pure FF were respectively allocated. And the rates of fertilization were almost similar in all groups as resulting 82.81% in control, 85.07% in 10% FF, 87.71% in 40% FF and 81.25% in pure FF. On the examination for embryonal cleavage from fertilized eggs, the rates of developing to 4 cell stage was similar in all groups, as results 98.11% in control, 98.27% in 10% FF and 98% in 40% FF but 78.84% in pure FF. And the rates of developing to 8-16 cell stage were significantly reduced as 44% in 40% FF and 44.23% in pure FF (p<0.05) compare to 71.69% in control media. As likewise, the rates of developing to morular stage were also significantly reduced to 36% (p<0.05) and 21.15% (p<0.01) respectively in 40% FF and pure FF. And the rates to blastocystic stage of embryo was lowest as 7.69% in pure FF (Table 1). The quality of embryonal cells on cleavage to the 8-16 cell stage was poorer, higher concentrations of FF. The rates of grade 1 in pure FF, as 23.07%, was lowest compare to those of other groups, in which the rates of grade 1 in control, 10% FF and 40% FF were 58.49%, 47.36% and 34% respectively. And on the contrary, the rate of grade 4 in pure FF was highest as 23.07%, while those were 5.66% in control, 8.77% in 10% FF and 20% in 40% FF (Table 2). On the viability of embryos, the rate of embryonal cell death was more rise, at the higher concentrations as well as longer exposure in the follicular fluid. At 48 hours after in vitro culture of embryos, the rate of survival embryos in pure FF was markedly lowered as 44.23%, compare to that of control (p<0.05). But there was not significant difference between the rates of survival embryos in each group beside the pure FF, which the rates were 77.35% in control, 70.17% in 10% FF and 60% in 40% FF respectively. And at 72 hours after in vitro culture, the rates of survival embryos were also significantly dropped to 21.15% in pure and 36% in 40% at concentration of FF compare to 62.26% in control (p<0.05, p<0.01). Finally, the rate of embryonal death at 96 hours after in vitro culture was highest as 82.69% in pure FF among all groups which those were 35.84 in control, 56.14% in 10% FF and 64% in 40% FF respectively (Fig. 1, 2, 3). In conclusion, this study suggests that the FF has no effects, in particular, to the in vitro fertilization of oocytes but exerted a bad effect to the cleavage, quality and viability of the embryonal cells during in vitro culture. However, the FF is harmful on embryonal development at conditions in higher concentration and especially on the embryos after $8{\sim}16$ cell stage.

• 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.

• Journal of Korean Society of Forest Science
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• v.108 no.4
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• pp.522-530
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• 2019

Two edible plants, Allium victorialis var. platyphyllum and Ligularia stenocephala, and one medicinal plant, Panax ginseng, were cultivated in the understory of an artificial Larix kaempferi plantation for ten years. Growth characteristics (number of leaves and flower stalks per plant, and leaf length and width), survival rate, and yield (fresh weight of plants) per unit area (1 ㎡) were investigated one year after planting, and six and ten years following cultivation. P. ginseng and L. stenocephala survived at a high percentage for two years after planting. Results showed that P. ginseng had longer and thicker roots when aged; however, a large number of plants died and the yield was low. In contrast, almost half of A. victorialis var. platyphyllum died within two years of planting. The surviving plants grew well for ten years and the yield was increased. The leaf length and width of L. stenocephala increased; however, the survival rate and the number of leaves per plant decreased as the period of cultivation was extended. In contrast, A. victorialis var. platyphyllum survived at a lower rate (50%) than the two other crops (98% for L. stenocephala and 83% for P. ginseng) during the early cultivation period, with little change in the survival rate over an extended time; however, the yields increased. This species showed an increase in the number of flower stalks and leaves, and as a result, the larger leaves increased the yield. We evaluated the understory cultivation of three crops in a L. kaempferi plantation under natural conditions, with no irrigation or fertilization, and Allium victorialis var. platyphyllum showed the greatest growth potential among the three tested crops.

• Clinical and Experimental Reproductive Medicine
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• v.25 no.2
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• pp.171-177
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• 1998

The combination of testicular sperm extraction (TESE) with ICSI can achieve normal fertilization and pregnancy rate and is effective method in obstructive and non-obstructive azoospermic patients. But, when pregnancy was not occurred, repeated testicular biopsies are not evitable. Therefore, in this study, we observed the survival rate of testicular spemratozoa and spermatozoa extracted from the seminiferous tubules after cryopreserved-thawed used for next IVF cycle with ICSI. In a total of 23 cases, obstructive azoospermia was 17 cases and non-obstructive azoospermia was 6 cases. In obstructive azoospermia, after thawing, motile spermatozua was observed in 13 cases (76.5%). The fertilization rate with 2PN was 67.6% and 5 pregnancies (29.4%) were achieved. In non-obstructive azoospermia, motile spermatozoa was observed in 2 case (33.3%) after thawing. The fertilization rates with 2PN was 53.7% and 3 pregnancies (50.0%) were achieved. A comparison of the results of motile spermatozoa after thawed testicular spermatozoa and spermatozoa extracted from the thawed seminiferous tubule section were 3 cases (60.0%) and 12 cases (66.6%), respectively. The fertilization and pregnancy rates of thawed testicular spermatozoa and spermatozoa extracted from the thawed seminiferous tubule section were 69.4% and 20.0%, 62.5% and 38.8%, respectively. Conclusively, thawed testicular spermatozoa and spermatozoa extracted from the thawed seminiferous tubule section can achieve normal fertilization and pregnancy and cryopreservation of testicular spermatozoa and seminiferous tubule may avoid repetition of testicular biopsies in azoospermic patients in whom the only source of spermatozoa is the testis.