The ovum pick up(OPU) technique can be used to produce embryos after in vitro culture of ovarian oocytes, can be used for early securement for effective herd early proliferation and excellent Hanwoo genetic resources, It is attracting attention as a very important technique for establishing technology. In addition to in vitro culture techniques, the number of oocytes retrieved depends on the environment and timing of the OPU. This study was conducted to compare and examine seasonal effect to the differences in the number of recovered oocytes, recovery rate and embryo development rate using Korean cattle kept in animal genetic resource research center by OPU technique. The grade of COCs was evaluated by microscopic examination. Grade A had 3 or more layers of cumulus cell and compact cytoplasm. Grade B had 1~3 layers of cumulus cell and compact cytoplasm. Grade C had 1 layers cumulus cell and compact cytoplasm. Grade D was denuded oocyte and poor cytoplasm. The recovery rate was 47.8±3.4% in summer (June to August) and 51.6±3.8% in autumn (September to October). The number of oocytes was 5.7±0.6 in summer and 5.2±0.7 in autumn. Oocyte grade A and B was 46.2%±6.3% in summer and 51.1±5.0% in autumn. The cleavage rate was 46.1±7.1% in summer and 65.0±11.3% in autumn. Blastocyst development rate was 19.9±9.4% in summer and 29.0±8.7% in autumn. There was no difference the recovery rate of oocytes and the number of embryos between summer and autumn. Cleavage rate and blastocyst rate of autumn was higher than summer. we will investigate to study the appropriate method for the production of Hanwoo embryos and the systematic comparison.

Four pluriparous Korean black goat does were superovulated with FSH and mated with fertile bucks. Anesthetized animals were placed in lateral recumbency, then size 8 Foley catheter was inserted into the uterus through the cervix under the vaginal speculum and the balloon was inflated to fix the catheter in the uterine body. The opposite end of the catheter was connected to a 3-way and a flushing medium was infused into the uterus. Modified Dubecco’s PBS with 1% FBS was used as the flushing medium. Four goats were allocated in two groups depending on the type of medium infusion into uterus. Injection group; the flushing medium was injected into uterus and the infused medium was collected by to-and-fro method using a syringe. Gravity-flow group; the flushing medium was allowed to enter the uterus by gravity flow by lifting the medium bottle and drained out of the uterus into a collecting tube. All four goats had catheter inserted through the cervix and uteri flushed successfully. The volume (recovery rate) of recovered medium varied considerably from 87 ml/200 ml (43.5%) to 148 ml/160 ml (92.5%). Nine embryos/ova in total were recovered from Gravity-flow group goats. Although the embryo recovery rate was low, the possibility of a transcervical embryo recovery in Korean black goat had been proven in this preliminary experiment.

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 day administration of FSH, 25 mg 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 GnRH at time of 1 insemination and embryos were recovered 8 days after the 1 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 19.11.40 with DEC method than 12.00.44 with conventional embryo collection method, respectively (p<0.05). Also, The average number of transferable embryos were significantly higher (p<0.05) as 15.81.72 with DEC method than 6.90.35 from conventional embryo recovery procedures. Meanwhile, number of recovered embryos and number of recovered transferable embryos following the number of flushing times until 6 flushing were significantly higher as 8.60.53 and 8.60.53 from 2 flushing time than other groups (p<0.05). No. of Ear. B stage embryos were significantly higher as 3.90.90 and 3.90.90 with 2 flushing time in total collected embryos and transferable embryos (p<0.05). Com M stage embryos were significantly higher as 3.71.00 in 2 flushing time and as 2.20.76 in 3 flushing time for recovered embryos (p<0.05). In transferable embryos, Com. M stage embryos were significantly higher (p<0.05) as 3.71.00 in 2 flushing time and as 2.20.76 in 34 flushing time, also. No. of degradation embryos was significantly higher as 2.20.72 in 5 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 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.

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 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 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 11.250.63, 12.50.65 and 11.750.48 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 6.250.48, 7.250.48 and 7.250.63 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 0.280.32 1 than 1.80.12 1, 1.750.10 1 from 3-way, 2-way methods (p<0.05). Also, the time required for recovering embryos by DEC methods was significantly lower as 272 min than 513, 452 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.

본 연구는 과배란처리에 의한 수정란이식 시 우수한 수정란을 다량 확보하고 이식 후 수태율 향상을 위하여 공란우 및 수란우에 bST처리가 수정란회수를 및 수태율에 어떠한 영향을 미치는가를 조사하고자 실시하였다. 공란우는 Folltropin-V와 PGF를 이용하여 과배란처리를 유도하여 12시간 간격으로 2 straw치 3회 인공수정을 실시하였다. 공란우와 수란우는 대조구와 bST처리구로 구분하였으며, bST(500 mg)처리는 발정발현 후 미근부에 근육주사

During the last three decades considerable advances has been made in goat embryo production and transfer technology. The Korean native black goat is the most useful domestic ruminant in this country for biological investigation and application because it has a lot of merits such as relatively short generation period(1 vs 2 year for a cow), easy of handling, well adaptation, high fertility, convenient and inexpensive. This article covers the methods of superovulation, estrus synchronization, embryo collection and transfer techniques, pregnancy diagnosis and subsequent pregnancy and kidding rates for the production of transgenic Korean native black goats. More than one hundred goat kids have been produced as a result of our transgenic goat project via microinjection of foreign gene into pronuclei, in vitro culture, transfer of various stages of fresh and frozen-thawed microinjected embryos into oviducts or uteri of recipient does. We have got two transgenic goats carrying a transgene targeting the expression of recombinant human granulocyte colony stimulating factor(hG-CSF) to the mammary gland so far. Since collection and transfer of embryos in this species is usually accomplished by laparotomy, exteriorization of the reproductive tract for surgical embryo collection leads to the formation of post-operative adhesions. Nonsurgical or laparoscopic technique to reduce adhesions from repeated surgeries has great advantages in improving embryo production and transfer especially from valuable donors. We will discuss this later.

During the last three decades considerable advances has been made in goat embryo production and transfer technology. The Korean native black goat is the most useful domestic ruminant in this country for biological investigation and application because it has a lot of merits such as relatively short generation period (1 vs 2 year for a cow), easy of handling, well adaptation, high fertility, convenient and inexpensive. This article covers the methods of superovulation, estrus synchronization, embryo collection and transfer techniques, pregnancy diagnosis and subsequent pregnancy and kidding rates for the production of transgenic Korean native black goats. More than one hundred goat kids have been produced as a result of our transgenic goat project via microinjection of foreign gene into pronuclei, in vitro culture, transfer of various stages of fresh and frozen-thawed microinjected embryos into oviducts or uteri of recipient does. We have got two transgenic goats carrying a transgene targeting the expression of recombinant human granulocyte colony stimulating factor (hG-CSF) to the mammary gland so far. Since collection and transfer of embryos in this species is usually accomplished by laparotomy, exteriorization of the reproductive tract for surgical embryo collection leads to the formation of post-operative adhesions. Nonsurgical or laparoscopic technique to reduce adhesions from repeated surgeries has great advantages in improving embryo production and transfer especially from valuable donors. We will discuss this later.

The aims of this study are 1) to test oocytes and embryos collected from in-vivo and 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 the procedures of Funahashi et al. Fertilized oocytes were cultured in 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, and 10% fetal bovine serum was added to the culture medium thereafter. Embryos were treated with 7.5g/ml cytochalasin-B for 30 min, centrifuged at 13,000 g for 13 min and then exposed sequentially to an ethylene glycol (EG) vitrification solution, aspirated into OPSs, and plunged/thawed into/from liquid nitrogen. In vivo embryos were surgically collected from three donors after Al. Forty-six embryos (18, 9 and 19 embryos, respectively) 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 received surgically 34(control), 188 and 184 embryos (derived from abattoir), respectively. Another three recipients were received nonsurgically 150, 100 and 150 embryos, respectively. All recipient sows exhibited delayed returns to estrus. To our knowledge, these results suggest that required an improved techniques, more vigorous embryos preparation and cleaner uterous condition(use gilt).