• Journal of Embryo Transfer
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• v.28 no.2
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• pp.121-125
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• 2013

This study was carried out to investigate the general characteristics of semen such as semen volume, pH, sperm motility and sperm concentration of the semen collected from Shih Tzu dogs (age of 24 to 48 months, weight of 4 to 8 kg) by using the method of digital manipulation of the penis. The effect of preservation temperature and time on motility of fresh semen was also investigated in the present study. Semen was collected for 16 times from 4 male Shih Tzu dogs by multiple ejaculations (four times ejaculation per dog). The average of semen volume, semen pH, sperm motility and sperm concentration of the second fraction containing small volume of the initial third fraction per ejaculation were $2.11{\pm}0.31$ ml, $6.25{\pm}0.07$, $97.59{\pm}1.03%$ and $2.05{\pm}0.14{\times}10^8$ cells/ml, respectively. Average semen volume per ejaculate, semen pH, sperm motility and sperm concentration of the first fraction from the ejaculation were $1.12{\pm}0.15$ ml, $5.99{\pm}0.14$, $16.09{\pm}6.18%$ and $5.16{\pm}2.03{\times}10^5$ cells/ml, respectively. Those of second fraction were $2.07{\pm}0.29$ ml, $6.36{\pm}0.13$, $97.31{\pm}1.36%$ and $2.15{\pm}0.30{\times}10^8$ cells/ml, respectively. Those of third fraction were $2.60{\pm}0.29$ ml, $6.63{\pm}0.08$, $95.72{\pm}1.61%$ and $6.03{\pm}1.83{\times}10^7$ cells/ml, respectively. Sperm motility was significantly higher at $17^{\circ}C$ preservation temperature than at $5^{\circ}C$ or $36^{\circ}C$ during preservation period except 1 h preservation (P<0.05). When preservation temperature was $17^{\circ}C$, sperm motility was $96.69{\pm}1.49%$ at 1 h, $91.38{\pm}1.90%$ at 6 h, $88.38{\pm}2.34%$ at 12 h, $78.13{\pm}4.58%$ at 18 h, $58.44{\pm}8.57%$ at 24 h and $29.56{\pm}5.06%$ at 30 h, respectively.

• Journal of Ecology and Environment
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• v.31 no.1
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• pp.9-15
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• 2008

Growth and reproduction of Palaemon serrifer were described and analyzed in a population inhabiting tide pools in warm temperate waters in Korea. The water temperature varied greatly in the tide pools, ranging from 8$^{\circ}C$ to 27.8$^{\circ}C$ Population structure and growth were investigated using size frequency distribution data collected from January to December 2003. Sex ratios fluctuated, but were almost equal during the breeding period. Growth was continuous and size increased gradually throughout the year. Adult females were larger and grew faster than males. von Bertalanffy growth parameters for a one-year sample of females and males were estimated as $L_{i\ddot{A}}$ = 11.32, K = 0.311, $t_0$ = -0.4115 and $L_{i\ddot{A}}$ = 8.36, K = 0.228, $t_0$ = -0.9693 respectively. Breeding was seasonal, starting in May, peaking in August, and finishing by the end of August. The species showed continuous production of successive broods. Laboratory observation showed that females with embryos near hatching had ovaries filled with vitellogenic oocytes ready for spawning. The reproductive output (effort) of each female (mean number of eggs: $552{\sim}1355$) was not high. The mean embryo volume, $0.078mm^3$, is relatively small, indicative of low energy allocation to each embryo. Recruitment of juveniles was closely linked to the breeding period, beginning in September.

• Journal of Embryo Transfer
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• v.30 no.1
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• pp.17-21
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• 2015

Alteration in ion channel or transporter expression levels affects cell volume which is produced by movement of water and ion across the plasma membrane. In particular, aquaporin (AQP) channels among ion channels play a crucial role in movement of water across the cell membrane. This study was performed to identify whether AQP expression is changed in bovine follicular cystic follicles using microarray, RT-PCR and Western blotting analyses. In microarray data, AQP4 expression was decreased, whereas AQP7 was increased in cystic follicles. Additional experiments were focused on the AQP7 expression increased in cystic follicles. The microarray data was confirmed by semi-quantitative polymerase chain reaction (PCR) and Western blot analysis. AQP7 mRNA and protein expressions were significantly increased in the cystic follicles (p<0.05). Application of estrogen ($10{\mu}g/ml$) to bovine ovarian cells showed a trend of increase in AQP7 expression. From these results, we suggest that the increase in AQP7 expression in cystic follicles may play an important role in movement of water in bovine ovary. In addition, AQP7, a aquaglyceroporin permeating water and glycerol, could be a good target in development of methods for the cryopreservation of bovine ovary.

• Journal of Embryo Transfer
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• v.13 no.3
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• pp.219-226
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• 1998

Enucleation of oocytes is an important limiting step for embryo cloning. We propose an enucleation technique based on the removal of chromatin after oocyte activation by aspirating the second polar body containing complemented chromatin. In a preliminary experiment to determine an optimal age of oocytes enucleation in rabbits, oocytes were enucleated at 15~20 hours post hCG. Recently ovulated oocytes were enucleated at a higher rate than aged oocytes. Microsurgical removal of the complemented chromatin in the second polar body was significantly more effective in enucleating than aspiration of a larger cytoplasm volume surrounding the first polar body of metaphase-arrested oocytes(96.8% versus 70.4%; P〈0.05). Moreover, compared with a nuclear transplantation protocol based on enucleation of metaphase-arrested oocytes and preactivated oocytes followed by treatment with 5 $\mu$M ionomycin for 5 min and 2 mM DMAP for 1 hr, there was no significant difference in the rate of blastocyst development. The ease with which modified technique can be performed is likely to render this technique widely useful for research and practice on mammalian cloning.

• Clinical and Experimental Reproductive Medicine
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• v.43 no.4
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• pp.247-252
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• 2016

Objective: Heparin can modulate proteins, and influence processes involved in implantation and trophoblastic development. This study aimed to assess the improvement of clinical pregnancy and implantation rates after local intrauterine injection of low-molecular-weight heparin (LMWH) in patients undergoing intracytoplasmic sperm injection (ICSI). Methods: A randomised case/control design was followed in women scheduled for ICSI. The study arm was injected with intrauterine LMWH during mock embryo transfer immediately following the ovum pickup procedure, while the control arm was given an intrauterine injection with a similar volume of tissue culture media. Side effects, the clinical pregnancy rate, and the implantation rate were recorded. Results: The pregnancy rate was acceptable (33.9%) in the LMWH arm with no significant reported side effects, confirming the safety of the intervention. No statistically significant differences were found in the clinical pregnancy and implantation rates between both groups (p= 0.182 and p= 0.096, respectively). The odds ratio of being pregnant after intrauterine injection with LMWH compared to the control group was 0.572 (95% confidence interval [CI], 0.27-1.22), while the risk ratio was 0.717 (95% CI, 0.46-1.13; p= 0.146). No statistical significance was found between the two groups in other factors affecting implantation, such as day of transfer (p= 0.726), number of embryos transferred (p= 0.362), or embryo quality. Conclusion: Intrauterine injection of LMWH is a safe intervention, but the dose used in this study failed to improve the outcome of ICSI. Based on its safety, further research involving modification of the dosage and/or the timing of administration could result in improved ICSI success rates.

• Journal of Embryo Transfer
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• v.12 no.3
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• pp.293-300
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• 1997

Supplementation of maturation medium with additional granulosa cells has beneficial effect on in vitro maturation of bovine follicular oocytes and their subsequent cleavage and development in vitro. However, maturation system using granulosa cells have some disadvantages that collection of granulosa cells is cumbersome and metabolic activity of the cells is variable according to ovarian cycle or follicular size. We hypothesized that bovine immsture oocytes matured without granulosa cell coculture can fertilize and develop normally if the medium volume per oocyte is reduced during in vitro maturation. Immature oocytes were matured for 24 hours in a TCM199 containing 10% fetal calf serum, anterior pitultary hormone (0.02 AU /ml Antrinⓡ) and estradiol with or without granulosa cells in vitro. In Group 1, 35 to 40 oocytes were matured in a well of 4-well plastic dish containing 500 $\mu$l of maturation medium and granulosa cells, and 9 to 10 oocytes were matured in a 50-$\mu$l drop of maturation medium without granulosa cells in Group 2. After maturation, oocytes were coincubated with sperm for 30 hours in a modified Tyrode's medium (IVF). Inseminated oocytes were cultured in a microdrop (30 $\mu$l) of a synthetic oviduct fluld medium (SOFM) containing BSA, Minimum Essential Medium essential and non-essential amino acids for 9 days. As a preliminary experiment, we investigated the beneficial effect of granulosa cells during maturation on subsequent cleavage and development using the same type of culturedishes (4-well dish). Granulosa cells could not increase embryo cleavage after fertilization but significantly improved (p<0.05) embryo development to expanding blastocyst (Table1 and 2). In Group 1, 68 and 80% of inseminated oocytes have cleaved at 30 hours and 2 days after IVF, respectively, which is similar (p>0.05) to the result of Group 2 (69% at 30 hours and 78% at 2 days after IVF). The oocytes in Group 2 showed 21 and 11% of developmental rates to expanding and hatching blastocysts, respectively, which was not significantly different (p>0.05) from those (20 and 10%, respectively) of oocytes in Group 1. In conclusion, it has been clarified that a microdrop culture system without granulosa cells for in vitro maturation can support bovine embryonic development to blastocyst in vitro as readily as a granulosa cell coculture system.

• Journal of Embryo Transfer
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• v.29 no.1
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• pp.59-66
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• 2014

Transcription activator like effector nucleases (TALENs) are artificial restriction enzymes generated by fusing a TALE DNA binding domain to a DNA cleavage domain which remove and introduce specific genes to produce transgenic animals. To investigate the efficient laboratory techniques for the injection of TALEN mRNA, pEGFP-N1 commercial plasmid were microinjected into porcine parthenogenetic and in vitro fertilization (IVF). In Experiment 1, to investigate injection time, compared 4 different time durations (2 hr, 4 hrs, 6 hrs & 8 hrs) after post activation of parthenogenetic embryos and after 6 hrs of co-incubation with sperms in IVF embryos. There were significant difference (P<0.05) in development to the blastocysts (4.4, 8.9, 3.9, 0.6%), GFP expression in blastocysts (1.3, 5.7, 2.3, 0.0%) which injected after post activation of 4 hrs compared with other 3 groups. IVF embryos after 2 hrs and 4 hrs injected were expressed GFP significantly higher than rest of two groups (P<0.05). In Experiment 2, compared development of 2 different concentrations ($20ng/{\mu}l$ and $50ng/{\mu}l$) of EGFP injection. There were significant difference (P<0.05) between two treatments which has higher cleavage (58.8 vs 41.9%), blastocysts development rate (13.0 vs 11.1%) and GFP expressed blastocysts (5.7 vs 0.0%) in $20ng/{\mu}l$ than the $50ng/{\mu}l$ in parthenogenetic embryos. In IVF embryos, only $20ng/{\mu}l$ injected embryos were expressed GFP (4.2%) after 7 days of incubation and 77.3 vs 64.7% of cleavage, 26.4 vs 23.5% development to blastocysts. In Experiment 3, three different volumes (5, 10 and 20 pl) were microinjected into porcine embryos to determine the most appropriate volume. Out of 3 groups, significantly higher development rates of cleavage (68.3, 58.0, 29.3%), blastocysts (11.7, 12.7, 0.5%) and GFP expressed blastocysts (2.9, 7.8, 0.0%) were shown in the 10 pl group (P<0.05). In conclusion, these results imply that $20ng/{\mu}l$ concentration, 10 pl of volume and injection at 4 hrs after post activation for parthenogenetic and 2~4 hrs after IVF, $20ng/{\mu}l$ concentration and 10 pl volume for IVF embryos were more effective microinjection conditions.

• Journal of Embryo Transfer
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• v.23 no.3
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• pp.161-166
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• 2008

The techniques for the collection, cooling and freezing of semen and artificial insemination of horses are not fully understood in Korea. We investigated the percentages of total motile (TM) and progressively motile (PM) sperms after the collection, cooling and freezing of stallion semen. The average volume of semen was 167 ml in Thoroughbred and 68 ml in Arab. The average numbers of spermatozoa in Thoroughbred and Arab were $104\times10^6/ml$ and $86\times10^6/ml$ respectively. The average percentages of TM and PM were 82.3% and 88.6% in Thoroughbred, and 61.4% and 82.6% in Arab, respectively. The average percentage of TM at 4 hr after cooling at $5^{\circ}C$ was significantly lower than that at 0 hr ($30.0\pm4.1%\;vs.\;78.0\pm2.5%,\;p<0.05$), but the percentage of PM was similar between 66.5 and 73.2% at 0, 1, and 4hr. The average percentage of frozen-thawed Thoroughbred semen frozen in MFR5 extender was 56.2%, which was significantly higher than that of the semen frozen in LE extender (average 32.9%, p<0.05). The percentage of TM in Arab was similar for semen frozen in MFR5 extender and LE extender (18.2% and 21.2%, respectively), but the percentage of PM was significantly higher in sperm frozen in MFR5 extender than in sperm frozen in LE extender (69.0% vs. 36.4%, p<0.05). Four mares were artificially inseminated by Thoroughbred frozen-thawed semen and one of them fertilized at 11 day after artificial insemination. In this study, the collection, cooling and freezing of equine semen were possible under domestic conditions.

• Journal of Embryo Transfer
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• v.25 no.1
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• pp.51-55
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• 2010

The relationships of scrotal circumference (SC) to semen characteristics and the conception rate (70 days-nonreturn rate) of artificial insemination in farm were studied with 137 heads of bull in Hanwoo. The average and range of SC were $38.27{\pm}3.90$ cm and 26.0~52.5 cm, respectively. Hanwoo bulls were classified with SC, divided into 34 cm below group, 34~39 cm group, and 39 cm over group. The 5,487 semen records of 43 heads of bull from July. 1. 2007 to June. 30. 2008. were used to determine the relationships between SC and semen characteristics. The semen concentration and total sperm number of each group were 11.18, 16.68, and $17.4{\times}10^8/ml$, and 69.83, 101.64 and $114.40{\times}10^8$/ejaculate. The bulls with 34 cm or more SC were higher than the bulls with 34 cm below in semen concentration and total sperm number (p<0.01). But between SC and semen volume have no significant relationship (p>0.05). The 9,862 mating records of 44 farm with 137 heads of bull were used to determine the relationships between SC and conception rate. The conception rate of 1st artificial insemination were 73.31, 74.16, and 77.33 % in each group. Also SC was positively correlated with pregnancy rate (r=0.12, p=0.17). These results indicate that SC correlates positively with semen characteristics, and maybe with pregnancy rate in Hanwoo.

• Journal of Embryo Transfer
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• v.23 no.4
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• pp.239-243
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• 2008

This study was conducted to investigate the effects of type of the sugar supplemented to the extender on the vigor, viability and intact acrosomal rates of frozen-thawed dog spermatozoa. The ejaculated semen was diluted with TRIS-citric acid extender containing 200mM TRIS, 73mM citric acid, 6% (v/v) glycerol, 20% (v/v) egg yolk, 1% (v/v) antibiotics (streptomycin/penicillin), 44 mM sugar, which was either glucose, fructose or glucose-fructose combination, and distilled water to make the final volume of 100ml. Extended semen samples were cooled at $4^{\circ}C$ for an hour, packaged in 0.25ml straws, equilibrated for 10 minutes in liquid nitrogen vapor, and frozen in liquid nitrogen. Samples were thawed by placing straws into $37^{\circ}C$ water for 120 seconds. After thawing, vigor, viability and intact acrosomal rates of frozen-thawed semen were compared according to type of sugar. No significant differences were observed between glucose and fructose groups. In addition, combination of the 2 sugars also did not show any significant differences in the vigor, viability and intact acrosomal rates. In conclusion, glucose and fructose were equally efficient as sugar supplements for freezing extender.