• Korean Journal of Animal Reproduction
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• v.14 no.1
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• pp.50-56
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• 1990

Bovine oocytes obtained from follicles(2~5mm) of ovaries after slaughter were cultured in TCM 199 medium with 10~20% heat-inactivated estrus cow serum(ECS) for 25~27 hr, at 39$^{\circ}C$ under 5% CO2 in air. At the end of culture period, some oocytes were stained with 1% acetoorcein and examined for the evidence of oocyte maturation. The remainder were used to assess the potential of in vitro fertilization(IVF) with frozen-thawed spermatozoa and subsequent development in media with or without bovine oviduct epithelial cell (BOEC) co-culture. The results obtained were summarized as follows ; 1. The maturation rate of oocyte in vitro in TCM 199 medium with 15% ECS group(76.3) was superior to 10% ECS group(68.3%) and 20% ECS group(64.5%). 2. The IVF rates of oocytes matured in vitro, and formation rate of male and female pronuclei were 63.6%(77/121) and 93.5%(72/77), respectively. The incidence of polyspermy was very low(2.4%). 3. Of 73 oocytes fertilized in vitro and cultured in TCM 199 medium with 10% fetal calf serum for 7 days, 41(56.3%) were cleaved over 2-cell and only 1(2.4%) was developed beyond the 16-cell stage. 4. Of 76 oocytes co-cultured with BOEC, 58(76.3%) were cleavaged and 23(39.7%) were developed to morula and blastocyst stage. The results of this study indicate that co-culture with BOEC deserved a positive effect on the IVF oocyte development through the 16-cell block.

• 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.

• Proceedings of the Korean Society of Embryo Transfer Conference
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• 2004.10a
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• pp.83-84
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• 2004

• Asian-Australasian Journal of Animal Sciences
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• v.15 no.3
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• pp.340-345
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• 2002

In this experiment, oocytes were collected from goat ovaries available in slaughterhouse by follicle puncture method. Morphologically culturable type of oocytes which having compact, multilayered cumulus granulosa cell complex and evenly granulated cytoplasm, was separated under a stereozoom microscope. Oocytes were washed thoroughly in maturation medium containing TCM-199, $1{\mu}g/ml$ estradiol-$17{\beta}$, 0.5 ${\mu}g/ml$ FSH, $100{\mu}g/ml$ LH, 3 mg/ml BSA and 10% estrus goat serum. Washed oocytes were cultured into maturation medium on granulosa cell monolayer. Culture plate was then kept into $CO_2$ incubator at $38{\pm}1^{\circ}C$, maximum humidity and 5% $CO_2$ for 18 h. After maturation the oocytes were washed thoroughly with maturation medium containing polyvinyl alcohol (PVA) without serum and BSA and further cultured for 12 h for secretory proteins of oocytes. PVA medium was collected, pooled and concentrated by 5000 cut off centrisart. Secretory proteins were separated on 12.5% SDS-PAGE. A total number of 3.41 oocytes per ovary were obtained and 2.17 culturable oocytes per ovary were cultured into maturation medium. After 18 h of maturation, 4,567 oocytes (1.82 oocytes per ovary) were further cultured into serum and BSA free PVA medium for its secretory proteins. Four secretory proteins of oocytes with approximately molecular weight of 45, 55, 65 and 95 kDa were obtained on SDS-PAGE in silver staining and three proteins with approximately molecular weight of 45, 55 and 65 kDa in Coomassie brilliant blue staining. In conclusion, four secretory proteins with approximately molecular weight of 45, 55, 65 and 95 kDa was obtained from in vitro cultured oocytes of goats.

• Korean Journal of Animal Reproduction
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• v.25 no.1
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• pp.9-17
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• 2001

To determine the effect of the characteristics of the corpus luteum (CL) and serum metabolites on pregnancy rate following embryo transfer in Hanwoo cow, recipients were synchronized the estrus with CIDR. in vivo or in vitro produced bovine embryos were transferred to synchronized recipients. The characteristics of the CL were checked by rectal palpation and ultrasound. Blood samples were collected from jugular vein at Day 7 and analysed progesterone and serum metabolites. The results obtained from this study were as follows; 1. Total 397 embryos were transferred to recipient and 121 heads out of them were pregnant. The pregnancy rate was 30.5% and slightly differences between farms. 2. There was not significantly different the pregnancy rates between the size of CL and the cavity CL. In the recipients which had more than 2ng/$m\ell$ of serum progesterone level, the pregnancy rates was in higher than others (46.6% vs. 24.4%, 15.0%). 3. The highest pregnancy rate was obtained at 90 to 110mg/㎗ in serum total cholesterol, 14 to 16mg/㎗ in BUN, and 70 to 80mg/㎗ in serum glucose, respectively, and these metabolites in blood could be used as the criteria in the selection of recipient at embryo transfer.

• Journal of Embryo Transfer
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• v.28 no.3
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• pp.237-241
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• 2013

It is generally accepted that chronic stress impairs female reproduction. It negatively affects ovarian function and the number of ovulated oocytes. Chronic stress lowers the number of retrieved oocytes. Ovarian follicular development is regulated by both pituitary-derived gonadotropins and intraovarian regulatory factors. The main corticosteroids are cortisol, cortisone, 11-deoxycortisol and corticosterone, cortisol being one of the most commonly used welfare and stress physiological indicator. In this study, we investigated the effect of cortisol level on progesterone patterns and ovulation in the dog. Cortisol and progesterone level of serum were analyzed by radioimmunoassay. The day of ovulation was considered as the day when serum progesterone concentration was 6.0~8.0 ng/ml. In vivo dog oocytes were collected by flushing oviducts of mixed-breed bitches at three days after ovulation. We classified dogs as having group 1 (cortisol level, 0 ${\leq}$ or < $2{\mu}g/dl$), group 2 (corisol level, 2 ${\leq}$ or < $4{\mu}g/dl$), group 3 (cortisol level, 4 ${\leq}$ or < $6{\mu}g/dl$) and group 4 (cortisol level, $6{\mu}g/dl$ ${\leq}$). The patterns of progesterone were not different in four cortisol groups. The average numbers of retrieved oocytes was not different in four cortisol groups. These results suggest that different cortisol levels on estrus dogs do not affect ovulation, number of ovulated oocytes and progesterone changes.

• Korean Journal of Agricultural Science
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• v.12 no.1
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• pp.55-61
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• 1985

This study was carried out to investigate reproductive traits and sex hormone levels by growth in Korean native goats. Serum levels of FSH, LH, prolactin, estradiol-$17{\beta}$ and progesterone were determined every 15 days from the 70 days of age to 190 days by radioimmunoassay. The age and weight at sexual maturity were 183.6 days and 14.3 kg; the length of estrous cycle and estrus period were 20.3 days and 36.7 hours, respectively. The gestation period, litter size and body weight at birth were 148.4 days, 1.4 head and 1.8 kg, respectively. The levels of serum LH were highest with 3.93 mIU/ml at 70 days of age, thereafter decreased gradually to 1.21 mIU/ml at 190 days of age. The concentrations of FSH in serum were below 1.25 mIU/ml throughout the experimental period. The levels of prolactin were lowest with 3.09ng/ml at 85 days of age and highest with 4.65 ng/ml at 175 days of age. The serum levels of estradiol-$17{\beta}$ were increased with age, thus highest with 7.95 pg/ml at 190 days of age. The serum progesterone concentrations were maintained low levels (below 1.0 ng/ml) throughout the experimental period.

• Proceedings of the Korean Society of Developmental Biology Conference
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• 2003.10a
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• pp.128-128
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• 2003

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$^{\circ}C$, and 10% fetal bovine serum was added to the culture medium thereafter. Embryos were treated with 7.5$\square$g/ml cytochalasin-B for 30 min, centrifuged at 13,000 ${\times}$ 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).

• Korean Journal of Animal Reproduction
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• v.7 no.2
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• pp.8-26
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• 1983

Various early pregnancy diagnostic methods have been developed in order to improve the reproductive efficiency in cow, mare, mule, sow, sheep, goat, dog, cat, rabbit, buffalo, camel, elephant, monkey, deer, lion, coipus and guinea pig. These methods include abdominal swelling, abdominal palpation, esturs cylce detection, Lupin test, gonadotropin assay, colostrum injection test, sperm motility assessment, cervical mucus viscosity test, Kaber chromagens method, estrogen test, A Scheim-Zond다 test, spectrophotometric detection of estrogen in urine and feces, boric acid crystraline formation test in urine, oxytocin injection test, diamino-oxidase test, PMSG HA test, behaviour test, Simolus iodine detection test, detection of tryptophane in urine, x-ray method, Cuboni and Lunaas method, vaginal biopsy method, Friedmann Schneider diagnostic method, electrode method, barium chloride detection method, ECG, Doptone method, ultrasound method, ultrasound scanning method, LDH method, rectal palpation method, CL palpation method, radioautography, serum creatine test, serum globulin test, chlormadine method, CAP method, Medata Do, pp.ers method, body fluid test, Plasma oCS detection method, ERIA, LHRH method, negative latex cogulation test and oestrone sulphate detection method. The most reliable methods with high a, pp.icability to farm animals such as sheep, mare, sow and cow are rectal palpation, ultrasound method and hormonal assay in blood and milk. However, they require complicated laboratory works for the early diagnosis of pregnancy and in most cases, the simple and economical methods which are described up to now need a long period of time after conception. Generally, it is possible to detect pregnancy after one estrus cycle, even though it varies depending on the species of animals. For improvement of the reproductive efficiency, it is required to develop a more accurate, economical, simple and early detectable method. It is anticipated that the result of a study on the detection method of EPF(early pregnancy factor) would be a, pp.icable to various animals within 6 hours after conception.

• Journal of Embryo Transfer
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• v.15 no.3
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• pp.263-270
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• 2000

The ovaries of 178 Holstein heifers or cows (heifer; 41, 1 parity; 72, 2$\leq$ parity; 65) on Day 6 or 7 (Day 0=day of estrus) were examined by transrectal ultrasonography. Diameter of corpus luteum (CL) and large follicle ( $\geq$ 10 mm), and luteal tissue area were determined by ultrasound system with a 5 MHB rectal probe. Blood samples were taken to progesterone analysis. After selection of recipients, frozen Holstein embryos were thawed and directly transferred to recipients non-surgically. The diameter of CL and luteal tissue area was greater (P<0.01) on Day 7 than on Day 6 in heifers, 1 parity or 2 $\leq$ parity cows, respectively, although progesterone concentrations were not different. The presence of fluid-filled luteal cavitied or multiple CL (2 or more) did not affect serum progesterone concentration. A large follicles were observed in 67.4% of heifers or cows and the average diameter was 14.1 mm. Greater luteal tissue area attributed higher pregnancy in heifers, but not in cows, although there were no difference on pregnancy rate according to progesterone concentration in heifers or cows. The pregnancy rate of recipients contained a large follicle at embryo transfer was lower than that of recipients not contained. These results show ultrasonic assessment of ovaries in Holstein recipients is a reliable tool to determine the follicle and CL for recipient selection.