• Korean Journal of Animal Reproduction
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• v.24 no.3
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• pp.281-288
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• 2000

This experiment was carried out to investigate the optimal activation condition for parthenogenetic development. In order to activate oocytes at 22 h post onset of maturation, the oocytes were subjected to 5 $\mu$M ionomycin(I) for 5 min ,10 $\mu$M calcium ionophore(Ca) for 5 min, 2 mM 6-dimethylamino-purine(DMAP) for 3 h and 10 $\mu\textrm{g}$/$m\ell$ cycloheximide(CH) for 6 h alone or in combination. The activated oocytes were cultured in modified CR$_1$aa at 5% $CO_2$, 5% $O_2$, 90% $N_2$. l. The cleavage rates after 48 h culture of oocytes treated with I, Ca, DMAP and CH were 12.7%, 14.1%, 28.9% and 22.9%, respectively. There was no blastocyst formation. 2. The cleavage rates after 48 h culture of oocytes treated with I ＋ DMAP, I ＋ CH, Ca ＋ DMAP and Ca ＋ CH were 96.9%, 82.1%, 93.1% and 34.7%, respectively. Developmental rates to blastocysts were 10.4%, 5.3%, 17.6% and 7.1 %, respectively. When oocytes were treated with Ior Ca followed by DMAP, the blastocyst formation rate was significantly higher than other groups(P <0.05). 3. According to single activation treatment, pronucleus formation rates were 5.4%, 3.6%, 28.3% and 28.8%, respectively, Whereas, all oocytes treated with the combined activation agents formed 100% pronucleus.

• Korean Journal of Animal Reproduction
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• v.26 no.1
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• pp.79-85
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• 2002

We investigated the optimal concentration and exposure time of cycloheximide(CHX) on development of activated porcine oocytes following electrical pulse(EP). After 42~44 h maturation, oocytes were treated with 0.1% hyaluronidase, and denuded cumulus cells by pipetting. Oocytes were stimulated by electric pulse (1.2 kV/cm, 30 $\mu$sec, 1 pulse) or incubated for 3, 5 and 7 h in cycloheximide (1, 5 and 10 $\mu\textrm{g}$/$m\ell$, respectively) following electric pulse, and cultured for 8 days. Cleavage rate of oocytes activated with 10 $\mu\textrm{g}$/$m\ell$ CHX following EP was significantly (P＜0.05) higher than those of 1 $\mu\textrm{g}$/$m\ell$ (86.8% vs. 74.4%). The developmental competence of oocytes incubated to 5 $\mu\textrm{g}$/$m\ell$ of CHX was significantly (P＜0.05) higher development to blastocysts (13.3%), compared with 10 $\mu\textrm{g}$/$m\ell$ of CHX (5.6%). When the oocytes were activated with 5$\mu\textrm{g}$/$m\ell$ CHX for 3, 5, and 7 h following EP, the cleavage rate of oocytes in 5 h group(86.6%) was significantly (P＜0.05) higher than that in 3 h group(73.2%). The developmental rate of oocytes to morula in 5 and 7 h groups(26.7% and 16.4%) were significantly (P＜0.05) high than that in 3 h group(14.5%). Matured oocytes were activated with electric pulse (EP) or electric pulse combined with cycloheximide (EP + CHX) and cultured for 8 days. The rate of cleavage and development to blastocyst (80.1% and 11.6%) of activated with EP group were similar to EP combined with CHX group. When activated with EP or EP combined with CHX, the mean cell number of blastocysts were less in the activated with EP (18.67$\pm$5.53) than in the activated EP combined CHX (20.71$\pm$6.16), but not significantly different. This results suggest that, when the porcine oocytes were activated with CHX following EP, the developmental rate of activated oocytes can be improved by treated with a concentration of 5 $\mu\textrm{g}$/$m\ell$ CHX for 5 h exposure time.

• Korean Journal of Fisheries and Aquatic Sciences
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• v.22 no.5
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• pp.266-280
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• 1989

Gonadal maturation of the Korean pomfrets, Pampus echinogaster (Basilewsky) and Pampus argenteus (Euphrasen) were histologically investigated based on the samples captured in the East China Sea from January 1987 to December 1988. Gonadosomatic index (GSI) of P. echinogaster began to increase from March, and reached maximum between May and July. It began to decrease from July and reached mini-mum between August and February. P. argenteus had a similar cycle, however, P. argenteus has higher values in April than P. echinogaster. Hepatosomatic index (HSI) were positively related to GSI. HIS of P. echinogaster and P. argenteus reached maximum in $April\~July$ and $April\~August$, respectively, Fatness coefficient of two Pampus species were low in the summer, and high in the winter. Ovary is of saccular structure, and testis is of lobular structure. From February, the early oocyte (ca. $100\mu$ in diameter grows) rapidly at the germinal epithelium of ovarian sacs. From March to April the oocytes grew up to cu $400\~500\mu$ in diameter. At this stage, the yolk globules are accumulated rapidly in the cytoplasmic layer. From May, the oocytes roached ca. $650\~850\mu$ in diameter, and they are spawned in $May\~July$. After spawning the residual follicles and remained ripe eggs degenerate. From February, spermatogonia grows into spermatocyte on the epithelium of the testicular lobuli. From May, spermatozoa appeared and spawning occurs. After spawning, the epithelium is thickened and the remained spermatozoa degenerate. Annual reproductive cycle of two Pampus species could be divided into four successive stages: Growing stage ($March\~April$), Mature stage ($April\~May$), Ripe and spent stage ($June\~July$) and Recovery and resting stage ($August\~January$). Absolute fecundity of P. echinogaster was $9,441\~135,294$, and that of P. argenteus was $50,678\~221,894$. Absolute fecundity of two Pampus species were positively related to body length and total weight. Relative fecundity was positively related to body length, while it was reversely related to total weight. The increasing rate of absolute fecundity of P. echinogaster was lower than P. argenteus. In P. echinogaster half of female and male reached first maturity at body length of $15.0\~$17.9cm and $12.0\~14.9cm$, respectively. All of females and males reached first maturity at body length of $18.0\~20.9cm$ and $21.0\~23.9cm, respectively. In P. argenteus all of females and males reached first maturity at body length of 18.6cm and 16.7cm$, respectively.

• Journal of Embryo Transfer
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• v.21 no.2
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• pp.163-168
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• 2006

The present study was carried out to investigate in vitro development and post-thawed survivability of bovine embryos according to different ovary transport temperatures. Bovine ovaries were collected at a local slaughterhouse and were transported at 4 different temperature categories to laboratory: $7{\sim}10^{\circ}C\;(T1),\;11{\sim}17^{\circ}C\;(T2),\;18{\sim}25^{\circ}C\;(T3)$ and above $26^{\circ}C$ (control group). The cumulus-oocyte-complexes aspirated from ovaries were in vitro matured, fertilized and cultured. The rates of maturation (to metaphase II), cleavage and development to blastocysts were compared among treatment groups. Furthermore, frozen-thawed blastocysts were in vitro cultured to compare the survivability among groups. The maturation rates in the T1, T2 and T3 groups ($60.0{\sim}68.2%$) were significantly lower than that in the control group (81.8%, p<0.05). The cleavage rates in the T1 and T2 groups (52.6 and 54.5%) were significantly lower than that in the control group (83.6%, p<0.05). However, there was no difference in the development rate to blastocysts among all groups ($27.9{\sim}33.0%$, p>0.05). The survivability of frozen-thawed embryos was significantly lower in the T1 group (46.2%) than those in the T2, T3 and control groups ($68.8{\sim}7.13%$, p<0.05). In conclusion, the results suggest that ovary transport temperature at $26^{\circ}C$ may be optimal for the better in vitro development and the survival of frozen-thawed embryos produced in vitro Furthermore, exposure of ovary to temperature below $10^{\circ}C$ during transport may significantly decrease both in vitro development and survivability of frozen-thawed blastocysts.

• Korean Journal of Animal Reproduction
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• v.27 no.3
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• pp.215-223
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• 2003

This study was to investigate the effects of fertilization time and culture medium of pig oocytes matured in-vitro by liquid boar sperm. The sperm rich fraction (30∼60 ml) was slowly cooled to room temperature (20∼23$^{\circ}C$) by 2 h after collection. Semen was transferred into 15 ml tubes, centrifuged at room temperature for 10 min 800 ${\times}$ g, and the supernatant solution was poured off. The concentrated sperm was resuspended with 5 ml of the LEN diluent to provide 1.0${\times}$10$^{9}$ sperm/ml at room temperature. The resuspended semen was cooled in a refrigerator to 4$^{\circ}C$. The medium used for oocyte maturation was TCM-199 supplemented with 26.19 mM sodium bicarbonate, 0.9 mM sodium pyruvate, 10 $\mu\textrm{g}$/ml insulin, 2 $\mu\textrm{g}$/ml vitamin B$_{12}$ , 25 mM HEPES, 10 $\mu\textrm{g}$/ml bovine apotransferrin, 150 $\mu$M cysteamine, 10 IU/ml PMSG, 10 IU/ml hCG, 10 ng/ml EGF, 0.4% BSA, 75 $\mu\textrm{g}$/ml sodium penicillin G, 50 $\mu\textrm{g}$/ml streptomycin sulfate and 10% pFF. After about 22 h of culture, oocytes were cultured without cysteamine and hormones for 22 h at 38.5$^{\circ}C$, 5% $CO_2$ in air. Oocytes were inseminated with liquid boar sperm stored at 4$^{\circ}C$ for 2 days after collection. Oocytes were coincubated for 1, 3, 6 and 9 h in 500 ${mu}ell$ mTBM fertilization media with 1.0${\times}$10$^{6}$ sperm/ml concentration, respectively. Thereafter, oocytes were transferred into 500 ${mu}ell$ NCSU-23, HEPES buffered NCSU-23, PZM-3 and PZM-4 culture media, respectively, for further culture of 6, 48 and 144 h. The rates of sperm penetration and male pronuclear formation were higher in the fertilization times for 6 and 9 h than in those for 1 and 3 h. The rates of cleaved oocytes were higher in the fertilization times for 6 and 9 h (85.0 and 84.6%) than in those for 1 and 3 h (61.1 and 76.8%). The percentage of blastocyst formation from the cleaved oocytes was highest in the fertilization time for 6 h (33.6%) than in that for 1, 3 and 9 h (11.4, 23.0 and 29.6%). Mean cell numbers per blastocyst were 32.9, 27.6, 26.3 and 24.4 in the fertilization times for 6, 9, 3 and 1 h, respectively. The rate of blastocyst from the cleaved oocytes and the number of cells per blastocyst were higher in HEPES buffered NCSU-23 culture medium than in NCSU-23, PZM-3 and PZM-4 culture media. In conclusion, we found out that liquid boar sperm stored at 4$^{\circ}C$ could be used for in-vitro fertilization of pig oocytes matured in-vitro. Also, we recommend the coincubation time of 6 h in 500 ${mu}ell$ TBM fertilization medium with 1${\times}$10$^{6}$ sperm/ml concentration and the HEPES buffered NCSU-23 culture medium for in-vitro fertilization of pig oocytes matured in-vitro.

• Journal of Embryo Transfer
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• v.30 no.2
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• pp.73-82
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• 2015

This study was designed to evaluate the possibility of increase through dairy female offspring's ratio by transfer of pre-selected transferrable blastocyst that was produced by pre-selected X-bearing semen with OPU derived oocytes. Elite dairy female cow is demanded strongly compared with male, the so called, farmer wants to produce only an elite female dairy offspring as a candidate female dairy cow for producing milk. In our study, we selected 2 elite dairy bull semen from National Agricultural Cooperative Federation to pre-select X-bearing semen and 5 elite dairy female cows as donor for collecting of OPU derived oocytes. OPU derived embryo production system was carried out an aspiration of immature oocytes from 5 donor cows 2 times per week, total 200 times for 2 to 7 months by an ultrasonographic guided follicular aspiration system and then produced in vitro-produced blastocysts by in vitro maturation, fertilization and culture. Dairy donor semen selected H-319, 320 bull in National Agricultural Cooperative federation was sorted X-bearing semen by flow-cytometer and frozen for using IVF with OPU derived oocytes. Donor cows were selected 5 elite dairy cows from Gyeongju Dairy Cow Community and then disease tests such as 4 kinds of disease before selecting was checked. Oocyte proportion of grade 1 to 3 from total collected oocytes was significantly lower in donor A and B than those in donor C, D and E (82.16 and 70.03% vs. 90.0, 91.78 and 93.57%), respectively (p<0.05). However, number of oocytes per session in donor A, C and E was significantly higher than those in donor B and D ($7.77{\pm}3.26$, $5.85{\pm}2.10$ and $7.03{\pm}2.14$ vs. $4.68{\pm}2.61$ and $5.21{\pm}1.97$ oocytes), but donor A was significantly higher than donor C (p<0.05). Development to blastocyst in donor B, C and E was significantly higher than those in donor A and D (31.0, 25.0 and 25.0% vs. 14.3 and 4.5%), but donor A was not different in donor C and E (p<0.05). Nine out of 10 blastocysts (90.0%) derived from OPU blastocysts were confirmed male embryos that was induced with Y-bearing semen to confirm sex ratio only. Total 96 blastocysts derived from female bearing semen were transferred into synchronized recipients and then confirmed 42 recipients (43.8%) pregnancy rate, 36 offspring (37.5%) and 91.7% female sex ratio (33 female vs. 3 male offspring). Taken together all data, elite dairy female offspring could be produced effectively by in vitro production system between pre-selected x-bearing semen and OPU derived oocytes that would be influential breeder in the breeding of dairy farm to increase effectively elite dairy offspring ratio as well as net income in the dairy farmer.