• Journal of Embryo Transfer
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• v.21 no.2
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• pp.137-146
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• 2006

The objective of this study was to examine the effect of donor cell types, the source of recipient oocytes and estrous synchronization on pregnancy and delivery rates of somatic cell nuclear transfer (SCNT) embryos in Korean native goats. Recipient oocytes were surgically collected after superovulation. Ear cells and fetal fibroblasts were collected and cultured in serum-starvation condition (TCM-199 + 0.5% FBS) for cell confluence. The zonae pellucidae of in vivo- and in vitro-matured oocytes were partially drilled using a laser system. Single somatic cell was transferred into the enucleated oocyte. The reconstructed oocytes were electrically fused with 0.3 M mannitol. After the fusion, embryos were activated by Ionomycin+6-DMAP. NT embryos were cultured in mSOF medium supplemented with 0.8% BSA at $39^{\circ}C$ in an atmosphere of 5% $CO_2$, 5% $O_2$, 90% $N_2$ for 12 to 20 hr. One hundred and two SCNT embryos were transferred into 20 recipients and pregnancy rate at days 30 was 20.0%. Of them, one developed to term and delivered 1 kid. Ear cells showed significantly higher fusion (63.8 vs. 26.5%) and pregnancy rates (20.0 vs. 0.0%) than those of fetal fibroblast (p<0.05). The recipients synchronized by CIDR showed significantly lower pregnancy rates compared to that of recipient in natural estrus ($0.0{\sim}25.0%$ vs. 100%) (p<0.05). Cloned kid was born from the recipient in natural estrus. For the synchronization of estrus between recipient and donor, there was no difference between treatments (${\pm}0$ vs. +12 hr) in pregnancy rate. The first healthy cloned kid (Jinsoonny) was produced by transfer of SCNT embryos derived from in vivo oocytes and ear cells into a recipient goat whose estrus was synchronized with the donor. These results imply that donor cells for nuclear transfer may affect the success rate, and the estrus synchronization between donor and recipient animals can also be important.

• Journal of Embryo Transfer
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• v.24 no.1
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• pp.33-37
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• 2009

Multiple ovulation and embryo transfer (MOET) has the potential to increase the rates of genetic improvement in cattle. Thus this study was performed to investigate several factors influencing in vivo embryo production in Holstein cattle under field conditions. The donors were superovulated with Folltropin-V and $PGF_2{\alpha}$ combination method. From Day 10 onward, donors were superovulated by i.m., twice daily, administration of 400mg Folltropin-V given in a series of decreasing doses over a 4-day period: on the first day, 3.5ml; on the second day, 3.0ml; on the third day, 2.0ml; and on the fourth day, 1.5ml (20ml in total, equivalent to 400mg of NIH-FSH-P1). Estrus was induced by i.m. administration of 25mg prostaglandin $F_2{\alpha}$ on the sixth and seventh of FSH treatment. Estrus detection was performed twice daily beginning 24h after the first prostaglandin $F_2{\alpha}$ injection. Donor cows were artificially inseminated 12 and 24 h after first standing estrus with semen from a proven Holstein sire. Embryos used in this study were recovered Day 7.5 of the cycle (Day 0: first standing estrus). From 195 superovulated dairy cows, 2,104 eggs were recovered, of which 1,172 were classified as transferable embryos based on morphological evaluation of quality. The results are summarized as follows: 1. The numbers of recovered and transferable embryos did not significantly differ among the capacity of milk production that were < 10,000kg/305days (group 1), $10,000{\sim}12,000\;kg$/305days (group 2) or > 12,000kg/305 days (group 3) (p>0.05, Table 1). 2. No differences in the numbers of recovered and transferable embryos were found among the donor's postparient days (p>0.05, Table 2). 3. Also, the numbers of recovered and transferable embryos of each superovulation seasons did not significantly differ among the four groups (p>0.05, Table 3).

• Development and Reproduction
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• v.4 no.1
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• pp.45-52
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• 2000

Membrane type matrix metalloproteinases(MT-MMPs) have been suggested to play an important role during structural remodeling of various tissue. Expression patterns of MT1-MMP and MT2-MMP mRNAs were investigated in oocytes, embryos, ovary and oviduct of mouse during their differentiation or periovulatory period using RT-PCR technique. Both cDNA products of MT1- and MT2-MMP of immature oocytes were barely discernable with a minimum amount but the expressions were distinct in mature oocytes regardless that they were matured in vivo or in vitro. MT2-MMP was not expressed by 2-cell embryos but was expressed by 4-cell stage embryos. From the morula stage untill hatched blastocyst stage, embyos showed intesnse expression of MT2-MMP with a sudden increase at blastocyst stage. While mouse ovarian tissues showed both expression of MT1- and MT2-MMP, there was no stage-specific difference throughout the estrous cycle. Mouse oviducts also exhibit constant amount of both MT1- and MT2-MMP expressions throughout periovulatory period, i.e., before or after ovulation. These observations lead to suggest that the differential expressions of maternal MT1- and MT2-MMP during meiotic resumption of mouse oocytes and embryonic expression of MT2-MMP particularly at blastocyst stage might play a role in the differentiation of mouse oocytes and／or embryos. The precise function of MT1- and MT2-MMP with regards to their participation in the remodeling of ovarian and oviductal tissues remains in a question.

• Reproductive and Developmental Biology
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• v.35 no.3
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• pp.233-238
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• 2011

This study was performed to investigate the FSH levels for superovulation procedure in Korean Native Cattle (Hanwoo). The effectiveness of 200 mg and 400 mg of FSH to initiate superovulation was examined in Hanwoo. Donors, at random stages of the estrous cycle, received a CIDR 7 days later, 200 mg FSH group was treated with 40, 30, 20, 10 mg FSH levels in declining doses twice daily by intramuscular injection fur 4 days. Also, 400 mg FSH group was treated with 80, 60, 40, 20 mg FSH levels. On the 3rd day administration of FSH, 25 mg $PGF_2$ ${\alpha}$ was administered and CIDR was withdrawn. Donors were artificially inseminated twice at 12 hr intervals. The donor cattle received 250 ${\mu}g$ GnRH at time of 1st insemination and embryos were recovered 8 days after the 1st insemination. As a results, average number of CL treated with FSH 200 mg was higher as $20.9{\pm}1.20$ than $15.8{\pm}0.63$ for donors treated with FSH 400 mg, respectively(p<0.05). Treated group of 200 mg FSH level increased (p<0.05) the number of embryos recovered per procedure compared to 400 mg FSH level ($18.2{\pm}1.18$ vs. $12.38{\pm}0.52$, respectively). When treatment of 200 mg FSH was performed, average transferable embryos/ova increased (p<0.05) to $14.1{\pm}1.12$ from $6.8{\pm}0.33$ of treated of 400 mg FSH. Group of 200 mg FSH increased (p<0.05) to $8.3{\pm}0.76$ from $2.0{\pm}0.26$ in morula stage compare to 400 mg FSH group. Mean of total early blastocyst and expanded blastocyst stage embryos was similar (p<0.05) between the 200 mg and 400 mg FSH levels group ($4.7{\pm}1.19$ vs. $2.9{\pm}0.18$ and $1.2{\pm}0.40$ vs. $1.9{\pm}0.17$). These results suggest that 200 mg FSH level-based superovulation protocol with CIDR may be effectively used fur production of superior embryos in Hanwoo. In other words, the less level of FSH may be effectively applied for Hanwoo (Korean Native Cattle), because Hanwoo was smaller body size than beef or daily cow.

• Korean Journal of Animal Reproduction
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• v.22 no.3
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• pp.287-292
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• 1998

The objective of this study was to test whether ZP drilling using a 1.48$\mu$m diode laser beam on mouse IVF embryos becomes effective the hatching and normal in vivo development, as a preliminary test for obtaining the additional proof that the 1.48$\mu$m diode laser could be used safely for human applications. The results obtained in this experiment were as follows: when the hatched rates of mouse embryos by laser ZP drilling according to the embryonic stage were examined until 72 hr (in case of blast tocyst: day 4 after IVF) or 120 hr (in case of 4-cell: day 2 after IVF) after treatment, the d data of laser drilled blastocysts (81.8%) was significantly higher than those of control (hatching blastocyst: day 4 after IVF) (54.2%) and laser drilled 4-cell embryos (45.5%) (p<0.05). When the effect of laser drilling on implantation rates following embryo transfer in day 3 synchronized pseudopregnant recipients was examined, the l laser drilled group (48.7%) was slightly higher than that of control group (43.6%). In addition, when the several pregnant mice delivered in two groups were analysed their chromosomal normality and tested reproductive ability, all p pups were presented normal chromosomal number (n=40) and showed normal growth and reproductive ability. Therefore, these results dem-onstrated that ZP drilling using a 1.48$\mu$m diode l laser can increase the embryo hatching and ind duce the normal pregnancy of mouse embryos.

• Reproductive and Developmental Biology
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• v.39 no.2
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• pp.29-36
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• 2015

Pigs are considered an ideal source of human disease model due to their physiological similarities to humans. However, the low efficiency of in vitro embryo production (IVP) is still a major barrier in the production of pig offspring with gene manipulation. Despite ongoing advances in the associated technologies, the developmental capacity of IVP pig embryos is still lower than that of their in vivo counterparts, as well as IVP embryos of other species (e.g., cattle and mice). The efficiency of IVP can be influenced by many factors that affect various critical steps in the process. The previous relevant reviews have focused on the in vitro maturation system, in vitro culture conditions, in vitro fertilization medium, issues with polyspermy, the utilized technologies, etc. In this review, we concentrate on factors that have not been fully detailed in prior reviews, such as the oocyte morphology, oocyte recovery methods, denuding procedures, first polar body morphology and embryo quality.

• Korean Journal of Animal Reproduction
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• v.16 no.4
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• pp.347-352
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• 1993

This study was designed to develop the in vitro culture systemof mammalian preimplantation embryos. We proposed mouse fetal fibroblast cells (MFFC) from 14∼15 day mouse fetus. Zygotes from superovulated female ICR mice were cultured 96 hrs in simple defined media (T6) or on the monolayer of MFFC. In addition, to evaluate the action of the co-culture of MFFC, various diluted superoxide dismutase antibody (SOD-Ab) was supplemented into the monolayer of MFFC and zygotes were cultrued in presence or absence of SOD-Ab. The developmental rates of zygotes were significantly increased in co-culture with MFFC compared to the control. The rates of zygotes to the 4-cell stage in media treated with EDTA were higher than those cultured in MFFC but the proportions of morula and blastocyst were not differ between EDTA and MFFC. Interestingly blastocysts in co-culture with MFFC possessed as many as blastomere as those developing in vivo, but blastocysts cultured with EDTA had significantly fewer blastomeres. In addition, the treatment of SOD-Ab suppressed the beneficial effect of MFFC. Therefore, our findings suggest that co-cultrue system using MFFC may have an advantage in the development of mouse zygotes as well as embryonic differentiation.

• Korean Journal of Animal Reproduction
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• v.17 no.3
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• pp.263-267
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• 1993

Transgenic animals have become an important tool in the basic and applied sectors of genetic and biomedical sciences. In particular transgenes provide clear-cut markers in the spatial and temporal analysis of developing embryos for the understanding of developmental mechanisms. For the long-term use of plasmid vector in a particular purpose it would be necessary to develop one's own vector system which can be properly expressed in eukaryotic system. Plasmids were constructed from ori region of pUC19 and early region of SV40 through various steps. LacZ gene coding for $\beta$-galactosides was fused to early gene of SV40 in translational in-frame. Poly(A) tailing site of SV40 was inserted at the 3' lacZ so that initiation, elongation and terminatin be controlled by SV40 transcription (pSS4). Biological function of the constructed pSS4 was demonstrated via microinjection of the plasmid into fertilized loach eggs and subsequent detection of $\beta$-galactosidase in developing embryos. The result indicate that the newly constructed pSS4 is functional in a eukaryotic system in vivo. Thus pSS4 may be used as an efficient tool for the study of embryogenesis and a basic carrier for various genes for animal transgenesis.

• Journal of Embryo Transfer
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• v.27 no.4
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• pp.237-243
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• 2012

This research was investigated the relationship between the number of the transferable embryos and estrus expression rate, BCS (Body Condition Score), which affect the nutritional state of the cow, in Holstein donor cows. CIDRs were inserted into the vaginas of twenty two head of Holstein cows, regardless of estrous cycle. Superovulation was induced using folliclar stimulating hormone (FSH). For artificial insemination, donor cows were injected with $PGF_{2{\alpha}}$ and estrus was checked about 48 hours after the injection. Then they were treated with 4 straws of semen 3 times, with 12-hour intervals. Embryos were collected by a non-surgical method 7 days after the first artificial insemination. When BCS was $$\leq_-$$2.5, the total number of collected ova was 7.3 + 1.9, which is significantly lower (p<0.05) than the numbers 15.4 + 2.8 and 15.4 + 2.1 that were obtained when BCSs were 2.75 and $$\geq_-$$3.0, respectively. Whereas the numbers of transferable embryos were 5.2 + 1.4 when BCS was $$\leq_-$$2.5, which was smaller than the numbers 6.0 + 2.1 and 8.5 + 1.8 that were obtained when BCSs were 2.75 and $$\geq_-$$3.0, respectively; however, the differences were not significant. As for estrus induction rate, the cow groups whose BCSs were 2.75 and $$\geq_-$$3.0 showed 100.0% and 95.0%, respectively. Whereas the cow group whose BCS was $$\leq_-$$2.5 showed 57.1%, and the differences were significant (p< 0.05). As for estrous expression rate, the cow groups whose BCSs were $$\leq_-$$2.5, 2.75 and $$\geq_-$$3.0 showed 100.0%, 100.0% and 85.7%, respectively; however, the differences were not significant. According to the result of this research, it is considered that the total number of collected ova and the number of transferable embryos will be affected by the nutritional state before and after in vivo embryo production and superovulation treatment, and that although the mechanism is not clear, poor stockbreeding management and nutritional level would cause the decrease of ovum recovery rate and the number of transferable embryos in high-producing cows. On the other hand, diverse researches on the superovulation treatment method that is suitable for high-producing Holstein donor cows would contribute to preventing ovarian cyclicity disorder, as well as to the early multiplication of cows with superior genes by increasing the utilization value of donor cows.

• Journal of Embryo Transfer
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• v.19 no.2
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• pp.155-163
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• 2004

The aims of this study are 1) to test oocytes and embryos collected from in-vitro to achieving the valuable protocol by culturing, vitrifying and thawing of oocytes/embryos, and 2) to transfer them to recipient, and finally have resulted in pregnancies from recipient females after surgical or nonsurgical transfer. In vitro maturation and fertilization were performed according to Funahashi et al (1994). Glucose-free NCSU 23 supplemented with 5 mM sodium pyruvate, 0.5 mM sodium lactate and 4 mg/ml bovine serum albumin for 2 days at $39^{\circ}C$, and 10% fetal bovine serum albumin was added to the culture medium thereafter. Embryos were treated with 7.5 ${\mu}g/ml$ cytochalasin-B for 30 min, centrifuged at 13,000 rpm for 13 min and then exposed sequentially to an ethylene glycol(EG) vitrification solution, aspirated into OPS, and plunged/thawed into/from liquid nitrogen. In vivo embryos were surgically collected from three dornors after AI for control group. Forty-nine embryos were washed 3 times in mPBS + 10% FBS, followed treatments : cultured, centrifuged, vitrified, recovered and transferred to recipients as in vitro prepared embryos. Three recipients were transferred individually with 100, 100 frozen embryos derived from abattoir and 34 fresh embryos by surgically, and another three recipients were transferred individually with 150, 150 frozen embryos and 100 fresh embryos by nonsurgically, respectively. all recipient sows exhibited delayed returns to estrus. To our knowledge, theses results suggest that required an improved techniques, more vigorous embryos preparation and substitute to gilt with cleaner uterous condition.