• Fisheries and Aquatic Sciences
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• v.4 no.1
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• pp.39-46
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• 2001

To examine the effect of copy number-dependent transgenic genotype on the expression of foreign gene, stable hemizygous and homozygous transgenic breeding line was established using artificial parthenogenesis. For this purpose, induced diploid gynogenetic transgenesis was optimized in mud loach (Misgurnus mizolepis) using UV-irradiated cyprinid loach (M. anguillicaudatus) sperm and thermal shocks. Optimum UV range for inactivation of cyprinid loach sperm was between 3,150 to $4,050\;ergs/mm^2$ The UV-irradiated sperm were inseminated into eggs from recessive color strain (yellow) or heterozygous transgenic mud loach containing CAT gene. Cold shock at $2^{\circ}C$ for 60 min, 5 min post fertilization successfully restored the diploidy of eggs inseminated with UV-irradiated sperm. Restoration to diploidy was confirmed by flow cytometry and gynogenetic status was verified by examining maternal exclusive inheritance of multi-locus DNA fingerprints, body color and transgenic marker. Putative isogenic transgenic fish clearly showed homozygous status at trans gene locus based on Southern blot hybridization and progeny testing. Further, such homozygous gynogenetic diploids revealed the increased levels of transgene expression, when compared to those of heterozygous (hemizygous) transgenic fish.

• Journal of Aquaculture
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• v.12 no.3
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• pp.167-173
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• 1999

Blocking the 1st mitotic cleavage was performed in mud loach (Misgurmus mizolepis) using UV-irradiated cyprinid loach (M. anguillicaudatus) sperm and ternal shocks Optimum UV range for inactivation of cyprinid loach sperm and thermal shocks. Optimum UV range for inactivation of cyprinid loach sperm was between 3,150 to 4,050 ergs/m$m^2$. Heat shock treatment ($41^{\circ}C$ for 3mins) with various treatment initiation times ranged from 22 to 50 min post insemination resulted wide range of success for induced gynogenesis. Best result was obtained when haploid egges were shocked at 28 min after insemination (corresponding to metaphase division of the 1st cleavage); 26% of total eggs inseminated were viable diploid gynogens. The hatching success and early survival of the both meiotic and mitotic gynogenetic groups were significantly lower than those of control crosses (P<0.05). Maternal origin of induced gynogenetic mud loach was verified by multi-locus DNA fingerprinting.

• Korean Journal of Animal Reproduction
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• v.18 no.3
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• pp.183-189
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• 1994

We examined early development in loach(Misgurnus mizolepis) embryos with parthenogenetic agents well-known in mammals. Female loach was superovulated with an intraperitoneal injection of 15 IU human chorionic gonadotrophin (hCG) per gram body weight. After 13 h of hCG injection, the oocytes were obtained from the abdomen. The oocytes were activated with 10% ethanol in tap water or fish Ringer's solution for 5, 10 and 15 minutes(eTW5, 10, 15 and eFRS5, 10, 15), respectively. The activation rates were 29% and 10% in eFRS10 and eFRS15, 5% and 6% in eTW10 and eTW15 by judging the cleaved blastomeres. Whereas, no parthenogenetic embryo was produced by tap water or fish Ringer's solution alone. The activation rate with the fish Ringer's solution was higher than that of tap water. No embryonic development was observed by calcium ionophore, A23187, at concentrations of 10, 20, 40 and 100$\mu$M when treated for 1, 2.5 and 5 minutes, respectively. The activation agents did not cause early development as in mammalian eggs. Therefore, the results suggest that fresh water fish may have a different egg activation pathway from that of mammals.

• Korean Journal of Animal Reproduction
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• v.16 no.2
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• pp.157-164
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• 1992

This stduy was carried out to obtain basic information available on ovulation, spawning, fertilization rate, hatching rate, and deformity rate after hCG injection in cyprinid loach, Misgurnus mizolepis. The results obtained in these experiments were as follows: 1. Ovulation and spawning occurred simultaneously and spawing was completed within 1 hour after ovulation. 2. More than 80% of fertilization rates appeared within 12 hours at 21$^{\circ}C$, 8 hours at $25^{\circ}C$, and 4 hours at 29$^{\circ}C$, respectively, following the onset of spawing. Afterwards, the fertilization rates of released eggs sharply decreased in three different water temperatures. 3. More than 70% of hatching rates appeared within 8 hours at 21$^{\circ}C$, 6 hours at $25^{\circ}C$, and 2 hours at 29$^{\circ}C$, respectively, following the onset of spawing. Afterwards, hatching rates of spawned eggs abruptly decreased in three different water temperatures. 4. The deformity rates of hatched larvae were high at $25^{\circ}C$, 8 hours following the onset of spawing. 5. Based on the developmental ability of oocytes, the optimum time of fertilization was 4 hours (stage 5) following the onset of spawing.

• Korean Journal of Ichthyology
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• v.24 no.2
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• pp.94-100
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• 2012

The spine loach, Cobitis hankugensis endemic to Korea were collected at the Nokdong River, Unbong-eup, Namwon-si, Jeollabuk-do during June 2007 for study its eggs development and early life history. The eggs were obtained by injecting gonadotropin (LHRH-1) to female and were fertilized by dry method in the laboratory condition. The matured eggs were separative, demersal and light yellowish with $1.29{\pm}0.07mm$ in diameter average. Under $23{\sim}25^{\circ}C$, the eggs were hatched in 45~52 hours after fertilization, and just hatching larvae were $4.5{\pm}0.24mm$ in total length. On 5 days after hatching, they were $6.6{\pm}0.13mm$ in total length and their yolk sacs were completely absorbed. From 25 days after hatching, they became the juvenile stage with $9.0{\pm}0.49mm$ in total length. On the 100 days after hatching, their external forms and band patterns were similar to those of adults with average $27.3{\pm}2.82mm$ in total length.

• Korean Journal of Ichthyology
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• v.11 no.2
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• pp.126-133
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• 1999

Ultrastructure of the zona radiata and the micropyle of ripe eggs in the rainbow trout (Oncorhynchus mykiss), catfish (Silurus asotus) and loach (Misgurnus anguillicaudatus) were examined by light, scanning and transmission microscopes. The egg micropyle of rainbow trout and catfish consists of a funnel-shaped vestibule and a tapered canal transversing the zona radiata. The micropyle of rainbow trout and catfish showed the similar structure with flat pit and long canal. The micropylar wall showed the clockwise spiral structure in rainbow trout. The micropyle of the loach showed the type with a hollow pit leading into a short canal and the micropylar wall showed the counterclockwise spiral structure. There are numerous and various size pores at the surface area around the opening in every experimented fish. Interconnecting ridges were observed in the unfertilized eggs of every fish.

• Korean Journal of Ichthyology
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• v.28 no.3
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• pp.156-163
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• 2016

Egg development and early life history of Korean southern spine loach, Iksookimia hugowolfeldi, were observed in the present study. Eggs were obtained after injecting females with 0.5 mL/kg of Ovarprim. Eggs were artificially fertilized using the dry method in the laboratory. Number of spawned eggs were $1,933{\pm}530per$ individual. Mature eggs were slightly adhesive with light yellowish coloring, and they measured $1.35{\pm}0.03mm$ in diameter. Hatching of the embryo occurred 56 h (50%) after fertilization at water temperature $25^{\circ}C$, and newly hatched larvae were averaged of $5.6{\pm}0.18mm$ in total length. At 5 days after hatching, larvae averaged $7.8{\pm}0.31mm$ in total length and their yolk sacs had been completely absorbed. Beginning at 15 days after hatching, the fish entered the juvenile stage and reached $13.2{\pm}0.87mm$ in total length. At 100 days after hatching, the band patterns and external form of juvenile were similar to those of adults, and they averaged $49.2{\pm}4.29mm$ in total length.

• Korean Journal of Ichthyology
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• v.31 no.1
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• pp.30-38
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• 2019

Egg development and early life history of the Korean endemic sand spine loach, Cobitis nalbanti, were investigated in the present study. Adult fish were sampled using spoon nets at the Yeongsan River in Seongam-ri, Bukha-myeon, Jangseong-gun, Jeollanam-do, Korea, June 2011. Eggs were obtained after injecting Ovarprim into females. Eggs were then artificially fertilized using the dry method in the laboratory. Mature eggs were transparent and slightly adhesive with light yellowish coloring, measured $0.99{\pm}0.03mm$ ($mean{\pm}SD$) in diameter. And number of spawned eggs were $1,527{\pm}410$ per individual. Hatching (50%) of the embryo occurred 52 hours after fertilization a water temperature of $25^{\circ}C$, and the average newly hatched larvae size was about $4.2{\pm}0.22mm$ in total length. At fifth day after hatching, the larval total length reached $6.0{\pm}0.34mm$ on average and their yolk sac had been completely absorbed. At fifteen day after hatching, larva entered the juvenile stage and reached $10.8{\pm}0.45mm$ in total length. At 100th day after hatching, the formation of Gambetta's zone of four dotted line was complete and juveniles were similar in general appearance to adults, and they averaged $41.1{\pm}2.95mm$ in total length.

• Korean Journal of Animal Reproduction
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• v.11 no.3
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• pp.170-180
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• 1987

This study was undertaken to determine the effects of HCG and trout pitutary on sexual maturation and ovulation in the Korean loach, Misgurnus anguillicaudatus. Sexual maturation was observed in the fish treated with 250＋125 IU HCG within 2-4 days following injections. Total ovary weight and GSI were significantly greater in the hormone-treated fish than in the control fish. Furthermore, in the hormone-treated fish body weight increased by about 4-9 percent in 2 days. The water content of the ovulated eggs of the fish treated with 250＋125 IU HCG was also greater than that in the control. Changes in both macroscopic & microscopic appearnce of Korean loach ovaries are describled, as well as changes in the gonadosomatic index. Oocytes pass through seven cytologically changes in the relative stages. By examining these stages, it was found that oogenesis occures in two broad phases. The previtellogenic phasebegins as a new oogonia continues to arise following spawning. These rapidly develop into early perinucleolus oocytes, which in turn develop into resting stage oocytes. Most oocytes remain in this stage when they develop into late perinucleolus oocytes. The vitellogenic phase begins as these late perinucleolus oocytes become transformed into early maturing oocytes through the accumulation of yolk. The cytoplasm completely fills with yolk as oocytes reach the late maturing stage. Shortly before spawning the final hyaline stage of developing is reached. Changes in the microscopic appearances of the ovaries were well correlated with changes in both gonadosomatic index and macroscopic appearance.

• Journal of Aquaculture
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• v.13 no.4
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• pp.359-362
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• 2000

Mitotic gynogenesis was induced in the far eastern catfish, Silurus asotus using UV-irradiated heterospecific sperm and cold shock treatment. Eggs were activated with the sperm of mud loach, Misgurnus mizolepis which has been irradiated with UV at dose of 9,000 ergs/$mm^2$. To determine the optimum duration required to prevent the first cleavage, a cold shock at 4$^{\circ}C$ with duration of 20, 30 or 40 min was applied to the eggs 50 min after activation. To induce diploidization of mitogenesis, the most effective protocol was to apply cold shock to 50-min old (after activation) eggs at 4$^{\Circ}C$ for 30min.