• Journal of Life Science
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• v.27 no.10
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• pp.1097-1103
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• 2017

For the artificial induction of the sexual maturation of Anguilla japonica, salmon pituitary extract (SPE) is continuously injected into females, and the eggs obtained from artificial sexual maturation are artificially fertilized with sperms and hatched. However, repeated injection of SPE in the abdominal cavity causes tremendous stress in females, which may prevent their complete sexual maturation and reduce the immune system function, ultimately resulting in death. In addition, the poor quality of the ovulated eggs can reduce the hatching and survival rate of larvae. In the present study, sexual maturation of females was induced by inserting an osmotic (OS) pump containing hormone analogs known to effectively induce sexual maturation into the abdominal cavity of female eels, and the effect of the OS pump on the induced sexual maturation was investigated. Our study results showed that the gonadosomatic index (GSI) was significantly higher in the fish subjected to SPE injection than those subjected to human chorionic gonadotropin (hCG), gonadotropin-releasing hormone analog (GnRHa), and methyl testosterone (MT) injections, either separately or in combination. In addition, a histological analysis showed that the oocytes in the SPE OS pump groups were more mature (entered the nuclear shift stage) than those in the other groups. These results suggest that an osmotic pump containing hormone analogs can be used to induce sexual maturation in female A. japonica artificially.

• Korean Journal of Ichthyology
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• v.25 no.2
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• pp.65-73
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• 2013

Egg development and early life history of the Korean spine loach, Iksookimia pumila was investigated to provide basic information regarding biological characteristics and restoration. Adult fish were sampled using spoon net in the Baek Stream, Sangseo-myeon, Buan-gun, Jeollabuk-do, Korea, 7 July 2010. Eggs and sperms were obtained from the females and male with Ovaprim injecting (0.5 mL/kg) and then fertilized using the dry method in the laboratory. Number of spawned eggs were 1,107 (352~1,440). Spawned eggs were slightly adhesive, light yellowish coloring and measured $1.3{\pm}0.04$ mm (mean${\pm}$SD) in diameter. Spawned eggs hatched out 52 (47~55) hours after fertilization at water temperature of $23^{\circ}C$, and newly hatched larvae an average were $4.7{\pm}0.14$ mm in total length. At 5 days after hatching, larvae averaged $7.1{\pm}0.20$ mm in total length and their yolk sacs had been completely absorbed. Beginning at 17 days after hatching, fish entered the juvenile stage and reached $11.0{\pm}0.50$ mm in total length. At 100 days after hatching, the band patterns and external form of juvenile fish were similar to those of adults, and they averaged $31.3{\pm}3.98$ mm in total length.

• Clinical and Experimental Reproductive Medicine
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• v.27 no.2
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• pp.165-172
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• 2000

Objective: In vitro fertilization (IVF) and a prolonging the time of culture may be helpful in establishing a viable pregnancy through a selection effect. Some embryos do not develop beyond the 4-cell stage and some may not develop to the blastocyst stage. We have evaluated the safety of SET and the outcomes of pregnancy. Methods: Sperms were treated with Ham's F-10 supplemented with 10% human follicular fluid (hFF). oocytes or fertilized oocytes were cultured in Dulbecco's Modified Eagle Medium (DMEM) with 10% or 20% hFF respectively. Up to five oocytes were inseminated with approximately 200,000 sperm cells/2 ml in each well. Fertilization was examined in the following morning and fertilized oocytes were co-cultured until embryo transfer. Vero cells for co-culture were prepared in Tissue Culture Medium - 199 (TCM-199) with 10% fetal bovine serum. At the two to four cell and blastocyst on day 2 and day 5, embryo and blstocyst grading were evaluated. Pregnancy rate was determined after transfer of human embryos at the two to four cell stage on day 2 (Group I) or subsequent transfer of embryos on day 2 and at the blastocyst stage on day 5 (Group II). For statistical analysis, Student's t-test and Chi-square (${\chi}^2$_test) were used. Results were considered statistically significant when p value was less than 0.05. Results: No differences was found in the fertilization between Group I (81.0%, 98/121) and Group II (81.8%, 180/220). In case of cleavage rate, no difference was found in Group I (95.9%, 94/98) and Group II (97.8%, 174/178). However, the rate of-clinical pregnancy was significantly higher (p=0.014) in Group II (66.7%, 12/18) than in Group I (26.3%, 5/19). Conclusion: The results of this study showed that SET is safe and effective, and significantly increases the pregnancy rate.

• Korean Journal of Ichthyology
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• v.26 no.2
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• pp.89-98
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• 2014

Eggs development and early life history of the endangered Korean dwarf loach, Kichulchoia brevifasciata (Teleostei: Cobitidae) was investigated to provide basic information regarding biological characteristics and restoration. Adult fish specimens were sampled using a spoon net at Geurnsan-myeon, Goheung-gun, Jeollanam-do, Korea from June to July 2011. Since, spawning characteristics were analyzed, and females were induced to spawn by injecting Ovaprim (0.5 mL/kg) and their eggs were artificially fertilized with sperms by the dry method in the laboratory. Total length of mature female were 46~76 mm with GSI $9.6{\pm}3.77%$, and total length of mature male was 42~52 mm with GSI $3.5{\pm}1.04%$. Sex ratio (♂/♀) was 0.10, and there were no secondary sexual characteristics. The number of mature eggs was averaged $60{\pm}28.7$ per female. The lemon yellow eggs were slightly adhesive $1.46{\pm}0.07mm$ in diameter. The embryo hatched approximately 66 h after fertilization at $25^{\circ}C$, and the hatched larvae were averaged $5.5{\pm}0.07mm$ in total length (TL). At 6 days after hatching, the larvae averaged $9.0{\pm}0.29mm$(TL) and their yolk sac was completely absorbed. At 17 days after hatching, they entered the juvenile stage and reached $12.6{\pm}0.24mm$ (TL). At 80 days after hatching, the band patterns and external form of the juveniles were similar to those of adults, and they averaged $33.0{\pm}2.19mm$(TL).

• Journal of Aquaculture
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• v.7 no.3
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• pp.151-158
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• 1994

To evaluate the reproductive ability of gynogenetic diploid male. Paralichthys olivaceus. histological analysis of testis. cytological analysis of spermatozoa and fertilization test with normal aggs were studied and the results are as follow; The gonads of gynogenetic diploid male were histologically normal. and many spermatozoa were observed in their testis. Number of spermatozoa from the control and gynogenetic diploid male were $2.58\times10^9$ and $2.42\times10^9$ cells per 1 ml of milt. respectively (P> 0.05). Amount of milt per kg body weight from the gynogenetic diploid male was significantly higher (P< 0.01) than that from the control male (8.3ml). Size and morphology from the two experimental groups were not different (P>0.05). More than $80\%$ of fertilization rates and hatching rates were observed when the eggs from the control were fertilized with the gynogenetic diploid male sperms.

• Journal of Aquaculture
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• v.7 no.3
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• pp.159-164
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• 1994

All-female diploid Paralichtlrys olivaceus populations were produced by the artificial fertilization with the normal eggs and gynogenetic diploid males sperms. All-female triploids were also conducted to the fertilized eggs by cold shock. Floating, fertilization and hatching rates of the all-female diploid eggs were not significantly different from that of the diploid control eggs (P>0.05). All-female triploids were also not significantly different from their diploid controls or all-female diploid groups (P>0.05) in the floating and fertilization rates of eggs. However, hatching rates of all-female triploid groups were lower than that of the control (P<0.05). Induction of the triploids were confirmed by the measurement of erythrocyte sizes and by the chromosome counts. The volumes of erythrocytes and nucleus of triploid were larger than those of diploids, respectively. Percent incidences of triploid were $92.6\%$ in this experiment. The chromosome number of diploids and triploids showed 2n=48 and 3n=72, respectively, and their karyotypes were consisted of all acrocentric chromosomes. The gonads of 4-month-old triploids were histologically sterile.

• Journal of Embryo Transfer
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• v.28 no.4
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• pp.373-379
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• 2013

Spermatogonial stem cells (SSCs) developed into sperms through spermatogenesis have been utilized as a useful tool in the field of regenerative medicine and infertility. However, a small number of highly qualified SSCs are resided in the seminiferous tubule of testis, resulted in developing effective in-vitro culture system of SSCs for solving simultaneously quantitative and qualitative problems. Presently, SSCs can be enriched on testicular stromal cells (TSCs), but there are no systematic researches about TSC culture. Therefore, we tried to optimize culture condition of TSCs derived from mouse with different strains. For these, proliferation and viability were measured and compared by culturing ICR outbred or DBA/2 inbred mouse-derived TSCs at 35 or $37^{\circ}C$. In case of ICR strain, primary TSCs cultured at $37^{\circ}C$ showed significantly higher proliferation and viability than those at $35^{\circ}C$ and significant increase of proliferation and viability in sub-passaged TSCs was detected in the $35^{\circ}C$ culture condition. Moreover, sub-passage of primary TSCs at $35^{\circ}C$ induced no significant effects on proliferation and viability. In contrast, in case of DBA/2 strain, significantly improved proliferation were detected in the primary TSCs cultured at $35^{\circ}C$, which showed no significant difference in the viability, compared to those at $37^{\circ}C$. Furthermore, sub-passaged TSCs cultured at $37^{\circ}C$ showed no significant differences in proliferation and viability, compared to those at $35^{\circ}C$. However, with significant decrease of proliferation induced by sub-passage of primary TSCs at $35^{\circ}C$, no significant effects on proliferation and viability were resulted from sub-passage of primary TSCs at $37^{\circ}C$. From these results, culture temperature of primary TSCs derived from outbred and inbred strain of mouse could be separately optimized in primary culture and subculture.

• Reproductive and Developmental Biology
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• v.37 no.4
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• pp.263-267
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• 2013

Pig producers have been shown keen interest of the number of spermatozoa in a semen dose since pig artificial insemination introduce. However, determining the minimal number of spermatozoa need per AI without detrimental effect on overall reproductive performances is not an easy question to answer. To increase the efficiency of semen utilization in pig AI, optimum number of spermatozoa per dose needed to determine. The objective of this study was to determine the reproductive performance and factors that affect on-farm application of low-dose semen insemination in sows. Data were collected from Darby Genetics AI studs from 4th of June to 7th of July, 2012 (n=401). The numbers of parturition were 84, 234 and 83 in sows inseminated with doses of $1.5{\times}10^9$, $2.0{\times}10^9$ and $2.5{\times}10^9$ spermatozoa in 100ml extender, respectively. There were no significant differences on reproductive performances such as gestation period, total born, total born alive, stillbirth and mummy in sows inseminated with different semen doses. The average number of born alive was 10.5, 11.0 and 10.4 from sows inseminated with $1.5{\times}10^9$, $2.0{\times}10^9$ and $2.5{\times}10^9$ sperms, respectively. Also, number of spermatozoa per dose did not affect litter size (p>0.10). There were no significant differences of maternal genetic line difference on gestation period, total number born, number born alive, born dead and mummy. The estimated correlation coefficients of the different semen doses with total number born, number born alive, born dead and mummy were r=-0.00, -0.01, 0.02 and 0.02, respectively. Taken together, the result of this study suggested that when semen was appropriately inseminated after induced ovulation, insemination with low-dose ($1.5{\sim}2.0{\times}10^9$) semen dose not adversely affect sow's fertility.

• Journal of Animal Science and Technology
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• v.54 no.4
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• pp.283-290
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• 2012

Cryopreservation induces sublethal damage to the spermatozoa, which leads to their reduced fertile life. The objective of this study was to investigate the effect of taurine, hypotaurine and trehalose as antioxidants on the function of the freezing-thawed sperm in Korean Jeju Black Bull. The semen was cryopreserved with tris egg yolk extendercontaining 7% glycerol and treated with 20mM taurine, hypotaurine and trehalose. Frozen-thawed sperms were evaluated for sperm motility, viability, membrane integrity, acrosome integrity and sperm penetration ability. The results were compared to semen cryopreserved in tris egg yolk extender containing 7% glycerol only as control. Frozen-thawed semen evaluation clearlyindicated that the addition of taurine or hypotaurine significantly improved (p<0.05) the motility and viability compared to control spermatozoa. Moreover, in membrane integrity, swollen sperm ratio was significantly increased (p<0.05) in taurine, hypotaurine or trehalose compared to control. In sperm acrosome integrity, F pattern ratio was increased (p<0.05) in hypotaurine among treatments, and AR pattern was significantly lowered (p<0.05) in taurine, hypotaurine and trehalose. In assessed sperm fertilizing ability, taurine, hypotaurine or trehalose significantly improved (p<0.05) the ratio of pronucleus formation and SFI. Finally, compared with the control, addition of taurine, hypotaurine or trehalose as an antioxidant to the freezing extender showed more positive effects on the frozen-thawed spermatozoa. It is concluded that the addition of taurine, hypotaurine, or trehalose to the freezing extender could reduce cryodamage of the Korean Jeju Black Bull spermatozoa.

• The Korean Journal of Malacology
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• v.26 no.3
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• pp.235-244
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• 2010

Ultrastructural characteristics of the testis and spermatogenesis of Crassostrea gigas were investigated by Transmission and Scanning Electron microscope observations. The testis is a diffuse organ consisting of branching acini containing differentiating germ cells in a variety of stages. The acinus is surrounded by an intermitent layer of myoepithelial cells andis divided into subcompartments that are partially separated by pleomorphic accessory cells which remain in close contact with germ cells until late stages of development. these accessory cells contain a large quantity of glycogen particles and lipid droplets in the cytoplasm. Therefore, it is assumed that they are involved in the supplying of the nutrients for germ cell development, while any phenomena associated with phagocytosis of undischarged, residual sperms by lysosomes could be find in the cytoplasm of the accessory cells. The morphology of the spermatozoon has a primitive type and is similar to those of other bivalves. Mature spermatozoa consist of broad, cap-shaped acrosomal vesicle, subacrosomal material (containing axial rod embedded in a granular matrix), a oval nucleus showing deeply invaginated anteriorly, two triplet substructure centrioles surrounded by four spherical mitochondria, and satelite fibres appear to the distal centriole and plasma membrane. Spermatozoa of C. gigas resemble to those of other investigated ostreids. In particular, the anterior region of the acrosomal vesicle is transversely banded. It is assumed that differences in this acrosomal substructure are associated with the inability of fertilization between the genus Crassostrea and other genus species in Ostreidae. Therefore, we can use sperm morphology in the resolution of taxonomic relationships within the Ostreidea. The spermatozoon is approximately $42-47{\mu}m$ in length including an oval sperm nucleus (about $0.91{\mu}m$ in length), an acrosome (about $0.42{\mu}m$ in length) and tail flagellum ($40-45{\mu}m$). The axoneme of the sperm tail flagellum consists of nine pairs of microtubules at the periphery and a pair at the center. The axoneme of the sperm tail shows a 9 + 2 structure. These morphological charateristics of acrosomal vesicle belong to the family Ostreidae in the subclass Pteriomorphia.