This study was conducted to investigate the effects of alpha-lipoic acid (aLA) as an antioxidant that decrease the reactive oxygen species (ROS) in bovine embryonic development. Slaughterhouse derived bovine immature oocytes were collected and 4 different concentrations (0, 5, 10 and 20 mM) of aLA was supplemented in bovine in Vitro maturation (IVM) medium. After 20 hrs of IVM, maturation rates, levels of ROS and glutathione (GSH), and further embryonic development after parthenogenetic activation (PA) and in Vitro fertilization (IVF) was investigated according to aLA concentrations. Maturation rate was significantly higher in 10 mM group than other groups (80.5% vs. 62.9, 73.9, 64.2%; P<0.05). In the levels of ROS and GSH in matured oocytes as an indicator of oocyte quality, significantly better results were shown in 5 and 10 mM groups compared with other 2 groups. After IVM, significantly higher rates of blastocyst formation were shown in 10 mM groups in both of PA (27.9% vs. 18.8, 22.3, 14.2%; P<0.05) and IVF (32.6% vs. 23.9, 27.3, 16.2%; P<0.05) embryos. In addition, significantly more cell total cell number and higher inner cell mass ratio in 10 mM PA and IVP blastocysts showed developmental competence in 10 uM groups. Therefore, based on the entire data from this study, using 10 μM of aLA confirmed to be the optimal concentration for bovine oocyte maturation and embryonic development.

The objective of this experiment was to explore the effects of Roscovitine (Rosco) prior to in vitro maturation (IVM) of immature pig oocyte. Brilliant cresyl blue test has been used to select the good quality of oocyte. Specifically, the effects of Rosco exposure on nuclear and cytoplasmic maturation, diameter, intracellular glutathione (GSH) and reactive oxygen species (ROS), and embryonic development after parthenogenetic activation (PA) and somatic cell nuclear transfer (SCNT), and gene expression levels in SCNT embryos have been measured. Cumulus oocyte complexes (COCs) have been exposed in 75 μM of Rosco for 22 and 44 h. The COCs that were matured in the IVM for 44 h without Rosco used as control group. Diameter of matured porcine oocytes 44 h culture with Rosco was significantly lower than 22 h culture with Rosco and control groups. GSH was higher in control group than 22 h and 44 h with Rosco but reduction of ROS in 22 h than 44 h with Rosco. In PA, exposure with Rosco 44 h oocytes group has been significantly lower than 22 h and control group in rates of maturation, cleavage and blastocyst formation. Similarly, in SCNT embryos rates of maturation, cleavage and formation of blastocyst have been also significantly lower in 44 h Rosco treated group than other two groups. SCNT embryos treated with Rosco 22 h showed greater expression levels of POU5F1, DPPA2 and NDP52Il mRNA compared with other two groups. Our results demonstrate that Rosco treatment with 22 h prior to IVM improves the development competence of porcine oocyte.

This study was conducted to establish the optimal chemical post-activation conditions in porcine embryonic development after parthenogenesis (PA) and somatic cell nuclear transfer (SCNT) using 4 different chemical compositions (cytochalasin B (CB), cyclohexamide (CHX), demecolcine (DC), 6-dimethylaminopurine (DMAP). Porcine embryos were produced by PA and SCNT and then, cultured for post-activation with CB (7.5 μg/mL), CB (7.5 μg/mL) + CHX (10 μg/mL), CB (7.5 μg/mL) +DC (0.4 μg/mL), and CB (7.5 μg/mL) + DMAP (2 mM). In PA embryonic development, cleavage rates have been significantly higher in CB group (94.7%) and CB+DMAP group (94.1%) than that of CB+CHX and CB+DC group (88.1 and 84.3%, respectively). There have been no significant differences in blastocyst formation rates among the four groups. In cell number of blastocyst was shown in CB group (42.3%) significantly higher than CB+CHX and CB+DC group (40.6 and 40.6%, respectively). In SCNT embryonic development, CB+DMAP group (89.7%) significant differences were found on embryo cleavage rates when compared with other three groups. Blastocyst formation rates in CB+DMAP group (26.9%) were significantly higher when compared with CB, CB+CHX, and CB+DC groups (25.5, 20.2, and 22.1%, respectively). In blastocyst cell number, CB+DMAP group (41.4%) was found higher significant difference compared with other three groups. Additionally, we have investigated survivin expression in early development stages of porcine SCNT embryos for more confirmation. Our results establish that CB group and CB+DMAP group for 4 h during post-activation improves pre-implantation improvement of PA and SCNT embryos.

As a part of the effort to improve post-transfer survival rate of embryos in Korean black goats, a technique for laparoscopic uterine transfer of blastocysts was carried out. A total of 26 transferrable embryos (morula to expanded blastocysts) were transferred to 13 recipient goats via transabdominal laparoscopic method. In consequence of our hormone protocol, 65% of the recipients (13/20) were found to have synchronized estrus. After confirmation of corpus luteum in each recipient goat, a Babcock laparoscopic forceps was inserted into the lower abdominal cavity to hold a uterine horn and fasten it near the peritoneum without causing injury. Then 7.5cm long 16G IV catheter was inserted directly into the uterine lumen through the abdominal wall. After removal of the stylet of the IV catheter, the embryo transfer tube (identical in size to the stylet and loaded with blastocysts) was inserted into the uterine lumen through the catheter to unload the embryos. Of the 13 estrus synchronized recipients, 9 were transferred blastocysts and 4 were transferred molurae (2 embryos in each recipient) in uterine ipsilateral to the ovary with corpus luteum. Four of the 9 recipients which blastocysts were transferred using this method has been confirmed pregnant (44.4% pregnancy rate).

The aim of this study is to evaluate the effects of vitamin or mineral supplements on the conception rates of dairy heifers when replacing the last injection of GnRH with hCG in ovsynch protocol (experiment 1) and also to investigate whether the estrus synchronization treatment in the heifer stage affects the conception rates after 1st parturition (experiment 2). In experiment 1, 50 heifers were randomly assigned into 3 groups: 20 heifers each in groups 1 and 2, and 10 in group 3. All three groups were treated with an intramuscular injection of GnRH on day 0 (day 0 = the day of program start), PGF2α on day 7 and hCG on day 9, and were inseminated on day 10, 12~16h after hCG injection. In group 1 (vitamin group), the heifers were treated with an intramuscular injection of 5 ml of vitamin-ADE 500Ⓡ, and group 2 (mineral group) was treated twice with an intramuscular injection of 30 ml of mineral supplement-LAPTOVETⓇ on a one-week interval beginning on the day of hormone treatment (day 0 and day 7 respectively). Group 3 (control) was treated only with hormones. Pregnancy diagnosis was performed by ultrasonography through a rectal probe. First service conception rates (FSCR) and average services per conception (ASPC) were recorded for all subjects. Of the total 50 heifers, 6 (2 in group 1, 3 in group 2, and 1 in group 3) heifers were eliminated due to accidents during experiment 1. FSCRs were 58.8% (10/17), 66.7% (12/18) and 44.4% (4/9) in groups 1, 2 and 3, respectively. ASPCs were 1.53±0.72, 1.27±0.59 and 1.63±0.74 in groups 1, 2 and 3, respectively. Although there were no significant difference between the groups, relatively good results (higher FSCR and lower ASPC) were obtained in both group 1 and 2. In experiment 2, 11 primiparous cows from group 2 of experiment 1 in heifer stage which had been treated both with the hormones for estrus synchronizing and mineral supplements (ES group), and 12 primiparous cows treated only with minerals (non-ES group) were compared to examine the effects of estrus synchronization program on conception rates after 1st parturition. Following the examination, postpartum ASPCs were 1.55±0.82 and 2.17±1.47 in ES group and non-ES group, respectively. The postpartum average days open (ADO) were 116±56 and 197±93 in ES group and non-ES group, respectively. Although there were no significant difference between the two groups, desirable results (lower ASPC and shorter ADO) were found in ES group after 1st parturiton. In conclusion, experiment 1 indicates that vitamin or mineral supplement with ovsynch protocol may have some positive effect on FSCR and ASPC of dairy heifers, and in experiment 2, ES program in heifer stage had a positive effect on ASPC and ADO following 1st parturition.

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.

Laparoscopic ovum pick-up (LOPU) is a convenient method for collecting oocytes in small ruminants. LOPU has the advantage of being a less invasive means of oocyte collection, thereby allowing for a repeated usage of the oocyte donor animals. A total of 25 Korean black goats were used in the winter season (December to February) and LOPU was applied to the goats which had been treated for superovulation more than two times during the last twelve months. Estrus was synchronized with an intravaginal insert containing 0.3 g progesterone for 10 to 12 days. Ovaries were hyperstimulated with eCG 1,000 IU oneshot, FSH with eCG (50 mg / 1,000 IU; 70 mg / 500 IU; 70 mg / 1,000 IU) oneshot or FSH multiple-shot with eCG oneshot (20 mg × 6 / 300 IU) given intramuscularly 72 h prior to LOPU. For these groups, the number of follicles (mean ± SEM) observed which developed to larger than 2 mm in diameter were 1.6 ± 2.5, 4.3 ± 3.1, 5.5 ± 4.2, 6.6 ± 2.1 and 8.8 ± 7.8, respectively. Oocytes were aspirated by using OPU needles and a vacuum pump. The overall oocyte retrieval rates were 41.4%. Oocytes were matured in TCM-199 supplemented with 10% (w/v) bovine serum albumin + 10 μg/ml FSH + 1 μg/ml 17β-estradiol for 27 h at 39℃ in 5% CO2 in air. Oocytes were parthenogenetically activated by ionomycin combined with 6-diethylaminopurine (6- DMAP). Total oocyte maturation and cleavage rate were 67.3% and 78.8%, respectively. In summary, LOPU is a useful oocyte collection method in Korean black goats that can provide immature oocytes for transgenesis or nuclear transfer.