• Journal of Embryo Transfer
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• v.24 no.4
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• pp.265-269
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• 2009

The purpose of this study was to determine the effect of different feeding ratios of whole crop barley silage on the embryo production in Hanwoo donors. All donors were basically fed 2.5 kg concentrate daily. Donors were divided into three groups according to the different feeding of forage; hay 70% and rice straw 30% (control, n = 21), whole crop barley silage 80% and rice straw 20% (T1, n = 25), and whole crop barley silage 60% and rice straw 40% (T2, n = 23) fed based on TDN 6.70/ BW 500 kg. All Hanwoo donors received a CIDR together with injections of 1 mg estradiol benzoate and 50 mg progesterone ($P_4$, Day 0). Four days later, they were superovulated with 28 mg FSH twice daily IM in decreasing doses over 4 days. Then donors received 2 doses of $PGF_2{\alpha}$ (25 and 15 mg) with the 5th and 6th injections of FSH on Day 6. CIDR were withdrawn at the 6th FSH injection and the donors received $100\;{\mu}g$ GnRH 36 h after the second $PGF_2{\alpha}$ injection. The donors were artificially inseminated twice, at 8 and 24 h after GnRH, and embryos were recovered 7 or 8 days after the 1st insemination. The flush rate of the donors following positive superovulation responses did not differ among groups (76.2~96.0%, p>0.05). The number of corpus luteum (CL) at embryo recovery also did not differ among groups (10.6~14.0, p>0.05). Furthermore, the mean numbers of total ova (9.4, 10.5 and 12.0) and transferable embryos (5.3, 12.0 and 6.5) did not significantly differ among the control, T1 and T2 groups, respectively (p>0.05). However, mean concentrations of serum $P_4$ of the T1 (64.2 ng/ml) and T2 groups (55.7 ng/ml) were higher than that of control group (43.3 ng/ml, p<0.01), while serum cholesterol concentrations in the control (105.8 mg/dl) and T2 groups ($96.9\;{\pm}\;mg/dl$) were significantly lower than in the T1 group (121.1 mg/dl, p<0.05). Conclusively, whole crop barley silage can be fed a good substitute for hay forage for Hanwoo donors. Furthermore the ratios of whole crop barley silage 60% and rice straw 40% might be more worthful for embryo production.

• Journal of Embryo Transfer
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• v.26 no.3
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• pp.159-164
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• 2011

This study was performed in order to determine optimum flushing solution using the direct embryo collection (DEC). Donors, at random stages of the estrous cycle, received a CIDR. 7 days later, 200 mg FSH was treated with 40, 30, 20, 10 mg FSH levels in declining doses twice daily by intramuscular injection for 4 days. On the 3$^{rd}$ day administration of FSH, 25 mg $PGF_2{\alpha}$ was administered and CIDR was withdrawn. After FSH injections were complete, donors were artificially inseminated twice at 12 hr intervals. The donor cattle received 250 ${\mu}g$ GnRH at time of 1$^{st}$ insemination and embryos were recovered 8 days after the 1$^{st}$ insemination. Embryo collection from superovulated donors were performed to flushing by DEC and conventional method. As a results, the average number of recovered embryos were significantly higher as 19.1${\pm}$1.40 with DEC method than 12.0${\pm}$0.44 with conventional embryo collection method, respectively (p<0.05). Also, The average number of transferable embryos were significantly higher (p<0.05) as 15.8${\pm}$1.72 with DEC method than 6.9${\pm}$0.35 from conventional embryo recovery procedures. Meanwhile, number of recovered embryos and number of recovered transferable embryos following the number of flushing times until 6${dr}$ flushing were significantly higher as 8.6${\pm}$0.53 and 8.6${\pm}$0.53 from 2$^{nd}$ flushing time than other groups (p<0.05). No. of Ear. B stage embryos were significantly higher as 3.9${\pm}$0.90 and 3.9${\pm}$0.90 with 2$^{nd}$ flushing time in total collected embryos and transferable embryos (p<0.05). Com M stage embryos were significantly higher as 3.7${\pm}$1.00 in 2$^{nd}$ flushing time and as 2.2${\pm}$0.76 in 3$^{rd}$ flushing time for recovered embryos (p<0.05). In transferable embryos, Com. M stage embryos were significantly higher (p<0.05) as 3.7${\pm}$1.00 in 2$^{nd}$ flushing time and as 2.2${\pm}$0.76 in 34$^{dr}$ flushing time, also. No. of degradation embryos was significantly higher as 2.2${\pm}$0.72 in 5${rd}$ flushing time, On the other hand, degradation embryos was not observed in transferable embryos (p<0.05). In conclusion, these results suggest that DEC method should effective methods for production of in vivo embryos using less flushing solution following perform until 4$^{rd}$ flushing time than conventional embryo collecting method. Also, it might be effectively collection of transferable embryos following more less procedure times compared to conventional embryo recovery methods.

• Journal of Embryo Transfer
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• v.19 no.3
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• pp.239-244
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• 2004

The present study was to investigate the pregnancy rate, gestation length and abortion rate of the recipients which transferred blastocysts produced by in-vivo collection, in-vitro fertilization (IVF) and nuclear transfer (NT). In addition, we investigate the birth weight and survival rate of the calves derived from the same methods. The pregnancy rate was 56.3％ in-vivo blastocysts, significantly higher than 19.4％ in NT blastocysts (p<0.05) but not significantly different from 30.0％ in IVF blastocysts. The abortion rate and the gestation length did not differ among the treatment groups (abortion rate: 0, 22.2 and 16.7％ respectively; gestation length: 278.8, 289.4 and 281.4 days respectively). The mean birth weight was significantly higher in NT calves (39.9kg) than in-vivo calves (25.5kg p<0.05). Recipients of in-vivo blastocysts (n=9) had all normal delivery and all of their calves survived on the 60$^{th}$ day from the birth. Recipients of IVF blastocysts (n=7) had all normal delivery but one of their calves died on the 48$^{th}$ day from the birth. Among recipients of NT blastocysts (n=5), three had normal delivery and two had Caesarean section. Among calves born through normal delivery (n=3) two died just after delivery but those born through Caesarean section all survived on the 60th day from the birth.

• Journal of Embryo Transfer
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• v.26 no.3
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• pp.153-158
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• 2011

This study was performed in order to simplify the operation and minimize stress of donor and be readily available in the field with low cost and high quality embryos using the Direct Embryo Collection (DEC). Donors, at random stages of the estrous cycle, received a CIDR. 7 days later, 200 mg FSH was treated with 40, 30, 20, 10 mg FSH levels in declining doses twice daily by intramuscular injection for 4 days. On the 3rd day administration of FSH, 25 mg $PGF_2{\alpha}$ was administered and CIDR was withdrawn. After FSH injections were complete, donors were artificially inseminated twice at 12 hr intervals. The donor cattle received 250 ${\mu}g$ GnRH at time of 1 st insemination and embryos were recovered 8 days after the 1st insemination. Embryo collection from superovulated donors was performed to flushing by non-surgical methods of 3-way, 2-way and DEC (l-way). The average number of recovered embryos were 11.25${\pm}$0.63, 12.5${\pm}$0.65 and 11.75${\pm}$0.48 from operations of 3-way, 2-way and DEC methods, respectively. There were no significant differences among the embryo collection methods. Also, The average number of transferable embryos were 6.25${\pm}$0.48, 7.25${\pm}$0.48 and 7.25${\pm}$0.63 from each embryo collection procedures. The number of transferable embryos was no differences among the 3-way, 2-way and DEC methods, respectively. Meanwhile, the ratio of transferable embryos for all recovered embryos from DEC methods was higher as 61.7 % than 55.6 %, 58 % from methods of 3-way, 2-way. And the flushing solution required for recovering embryos by DEC method was significantly lower as 0.28${\pm}$0.32 1 than 1.8${\pm}$0.12 1, 1.75${\pm}$0.10 1 from 3-way, 2-way methods (p<0.05). Also, the time required for recovering embryos by DEC methods was significantly lower as 27${\pm}$2 min than 51${\pm}$3, 45${\pm}$2 min, respectively (p<0.05). In conclusion, these results suggest that DEC method for embryo collection may be effectively used for production of in vivo embryos using less flushing solution and, it might be effectively available in the field compared to conventional embryo recovery methods using 3-way or 2-way balloon catheter.

• Proceedings of the Korean Society of Applied Pharmacology
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• 2003.11a
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• pp.87-87
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• 2003

Taurine (2-aminoethanesulfonic acid, $^{+}NH_3CH_2CH_2{SO_3}^{-}$) is endogenous $\beta$-amino acid which is essential in fetal nutrition and development and is present in abundant quantities in several tissues of fetus. In utero, taurine deficiency causes abnormal development and abnormal function of brain, retina, kidney and myocardium. Thus, transfer of taurine into fetus is important during embryonic development. Taurine transporter (TauT) has 12 hydrophobic membrane -spanning domains, which is typical of the $Na^{+}$- and $Cl^{-}$-dependent transporter gene family. Among the various biosynthetic enzymes of taurine, cysteine sulfinic acid decarboxylase (CSD) is the rate-limiting enzyme for biosynthesis of taurine. However, the enzyme activities of taurine biosynthesis are limited in early stage of embryonic development. To analyze the expression period of TauT and CSD during embryonic development, we have investigated the gene expression of TauT and CSD using reverse transcriptase polymerase chain reaction (RT-PCR) in mouse and chicken embryos. RT-PCR anaylsis revealed that both TauT and CSD mRNAs were already expressed at Day-4.5 in mouse embryo. In chicken whole embryo, TauT and CSD mRNAs began to appear on developing times of 48 hrs and 12 hrs, respectively. TauT mRNA was detected in the organs of heart, brain and eye of the day-3 chicken embryo. Our data show that TauT and CSD mRNAs were expressed in early stage of embryonic development.

• Journal of Embryo Transfer
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• v.26 no.2
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• pp.105-109
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• 2011

Ovulation synchronization (ovsynch) has proved to increase the number of insemination in cattle by overcoming the problems of heat detection. The aim of this study was to do ovsynch in water buffaloes where heat detection is a major reproductive problem and to determine the conception rates after timed artificial insemination (TAI). Twenty cyclic buffaloes at ${\geq}$ 60 days postpartum were selected by examining 24 unobserved estrus buffaloes based on milk progesterone assay (progesterone concentration ${\geq}$ 1.0 ng/ml) from the Mymensingh district of Bangladesh. Ovsynch treatment regimen was started irrespective of the stage of estrous cycle. Gonadorelin (500 ${\mu}g$) was injected intramuscularly at Day 0 followed by Alfaprostol (8 mg) at Day 7. A second injection of Gonadorelin was given at Day 9 and TAI was done with frozen semen from Mediterranean buffalo bulls at 16~20 hours of the second Gonadorelin injection. Milk progesterone ELISA at Day 10~12 post AI confirmed ovulation in 16 out of 20 (80%) buffaloes (progesterone concentration ${\geq}$ 1.0 ng/ml). High progesterone concentration (${\geq}$ 1.0 ng/ml) at Day 10~12 and Day 22~24 of AI showed pregnancy in six out of 20 (30%) buffaloes. Pregnancy was further confirmed by ultrasonography at Day 40 in these six buffaloes. In conclusion, ovsynch followed by TAI could be applied in cyclic buffaloes for overcoming the estrus detection problems; however, more studies are needed to increase the conception rate.

• Toxicological Research
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• v.29 no.1
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• pp.27-34
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• 2013

This study was conducted to investigate the potential embryo-fetal toxicity and toxicokinetics of the antimalarial agent artesunate (ARTS) in Sprague-Dawley rats. Pregnant rats were administered ARTS daily from gestational day 6~15 via oral gavage, at test doses of 0, 2, 4, or 8 mg/kg (22 females per group). The fetuses were examined for external, visceral, and skeletal abnormalities on gestational day 20. With regard to the dams, there were no deaths, treatment-related clinical signs, changes in body weight, or food intake in any of the treatment groups. There were no treatment-related gross findings at necropsy in any treatment group. In the 8 mg/kg group, there was a decrease in gravid uterine weight and in the weight of female fetuses. There was also an increase in fetal deaths (primarily late resorptions) and an increase in post-implantation losses (37%) at 8 mg/kg. An increase in the incidence of visceral and skeletal variations at 4 and 8 mg/kg was observed. These defects included minor changes in the appearance of the kidney and thymus, as well as absent ribs or thoracic vertebrae. Toxicokinetics were assessed in a parallel study, using 4 mated females per group. Using liquid chromatography-mass spectrometry (LC-MS) analysis, the concentration of ARTS and its metabolite dihydroartemisinin (DHA) were quantified in plasma from rats on gestational days 5, 6, 10, and 15. Amniotic fluid was assayed for ARTS and DHA on gestational day 15. There was evidence of rapid conversion of ARTS to the metabolite DHA in maternal plasma, since ARTS could not be consistently detected in plasma at the three doses tested. ARTS and DHA were not detected in amniotic fluid at gestational day 15, indicating limited placental transfer of the two agents. The embryo-fetal no-observable-adverse-effect level (NOAEL) of the test item was considered to be 8 mg/kg/day for dams, and 2 mg/kg/day for embryo-fetal development.

• Journal of Embryo Transfer
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• v.10 no.2
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• pp.131-138
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• 1995

Essential and non-essential amino acids supplemented to culture medium stimulate mammalian embryo development in vitro. Amino acids such as glycine, taurine and alanine are concentrated in the lumen of oviduct and uterus and it can he thought that these amino acids may have physiological role on fertilization and embryo development. Our aim of this experiment was to investigate the effects of essential and non-essential amino acids, taurine or glycine supplemented to fertilization medium on the cleavage and subsequent in vitro development of bovine oocytes matured and fertilized in vitro. Immature oocytes were obtained from slaughtered Holstein cows and heifers and matured in TCM199 containing 10% fetal calf serum, 2.5 $\mu$g /mL of FSH and LH and 1 $\mu$g / mL of estradiol with granulosa cells in vitro. After maturation, oocytes were coincubated with sperm in fertilization medium supplemented with Minimum Essential Medium (MEM) essential and non-essential amino acids, taurine (3.75 mM) or glycine (10 mM) for 30 hours in vitro. Inseminated oocytes were cultured in synthetic oviduct fluid medium (SOEM) containing MEM essential, non-essential amino acids and 1 mM glutarnine up to 8 days after fertilization.Supplementation of fertilization medium with MEM essential and non-essential amino acids lowered significantly (p<0.05 and p<0.001) the cleavage rate after 30 hours of IVF (53.3%) and at Day 3 (62.7%: Day 0: the day of I VF) compared to control (64.3% and 77.3%, respectively). Subsequent developmental rates to morulae (Mo) and expanding blastocysts (ExBL) also significantly decreased (p<0.001 and p<0.05 for Mo and ExBL) when oocytes were coincubated with sperm in the medium containing MEM amino acids. Taurine added to fertilization medium have not increased the cleavage rate over the control, whereas glycine showed significantly lower (p<0.01) cleavage rate at Day 3 than that of taurine, but there was no significant difference in the developmental rates to Mo and ExBL of bovine embryos irrespective of the supplementation of taurine or glycine to fertilization medium. In conclusion, supplementation of fertilization medium with essential and non-essential amino acids, taurine or glycine has no beneficial effect on in vitro cleavage and development of bovine oocytes matured and fertilization in vitro.

• Korean Journal of Animal Reproduction
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• v.19 no.2
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• pp.89-93
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• 1995

In this study was demonstrate that retrovirus-mediated gene transfer is one of the promising alternatives to the conventional pronuclear DNA microinjection approach, especially in transferring the exogenous genes into the boving embryos. By co-culturing of zona of zona-free one-cell stage embryos with the retrovirus-producing cells for 24 hours followed by 6 days of culture in virus-free medium, we could get morulae and blastocysts expressing the E. coli LacZ genes which were transferred by our retrovirus vector. The results obtained in this study are summarized as follows : 1. Addition of 5$\mu\textrm{g}$/ml of polybrene in the embryo and virus-producing cell co-culture medium did not affect development of zona-free one-cell embryo. 2. Compared with the intact embryos removal of zona at one-cell stage before co-culturing with the virus-producing cells for one day caused only slight decrease of embryo develpment. 3. Co-culture of 625 zona-free one-cell stage embryos with the virus-producing cells resulted in 65(10.4%) morulae or blastocysts, and 12.3%(8/65) of the morulae or blastocysts were E. coli LacZ positive.

• Journal of Embryo Transfer
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• v.23 no.1
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• pp.1-4
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• 2008

This study was performed to investigate the effect of laser-assisted hole in the zona pellucida (ZP) of frozen-thawed ICR mouse embryos on the process of hatching that is critical for expanded blastocysts to implant into endometrium, Vitrification medium, composed of ethylene glycol and sucrose supplemented with 7.5% (w/v) PVP, was used to freeze $2{\sim}4$ cell stage embryos recovered from oviducts of superovulated and mated female mice before storing them in $LN_2$. Right after thawing them, a laser beam was shot to make a hole in ZP followed by culturing in KSOM for $96{\sim}120\;hr$ and examining development to blastocyst and hatching every 12 hr. Laser-treated embryos showed significantly higher hatching rate compared to control (92.9% vs. 22.1%, p<0.05). From around Day 4, blastocysts developed from laser-treated embryos started hatching while the blastocysts of control group failed to hatch showing a lot of shrinkage. This study shows that a laser-assisted hole in ZP improves the hatching rate of blastocysts developed from frozen-thawed, in vitro cultured ICR mouse embryos.