• Journal of Embryo Transfer
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• v.17 no.2
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• pp.153-162
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• 2002

In order to develop the breeding techniques to increase Hanwoo (Bos taurus coreanae) population, the present study was performed 1) to establish the treatment protocol on reproductive disorders with GnRH or PGF／sub 2$\alpha$／, 2) to improve intraovarian PG $F_{2}$$\alpha$／ administration for reducing open period. Among total of 43 diagnosed, high percentage of cows (41.9％, 18 cows) were diagnosed as silent heat, followed by inactive ovaries (32.6％, 14 cows), ovarian cysts (9.3％, 4 cows), persistent corpus luteum (7.0％, 3 cows), endometriosis (4.7％, 2 cows), pyometra (2.3％, 1 cow) and luteal cysts (2.3％, 1 cow). To treat silent heat, 18 cows were administrated with 25 mg PGF／sub 2$\alpha$／, heat-detected, artificially inseminated and monitored pregnancy. All treated cows were heat-detected and 16 cows (88％) were successfully pregnant. With 200 $\mu\textrm{g}$ GnRH treatment, 7 cows (70％) with inactive ovaries and 3 cows (75％) with ovarian cysts were successfully pregnant. Administration with 25mg PGF／sub 2$\alpha$／, successfully treated 3 cows (100％) with persistent corpus luteum and 1 cow (100％) with luteal cysts, followed by 100％ pregnancy rate. With the combined treatment of 25 mg PG $F_{2}$$\alpha$／and antibiotics, 2 cows (100％) with endometriosis were effectively treated and got pregnant after. artificial insemination (AI). In order to reduce open period, 5 mg PGF／sub 2$\alpha$／ was administrated intraovarian to 20 days cows after delivery, heat-detected, artificially inseminated and monitored pregnancy. In the first experiment, in order to recover uterus, 5mg PGF／sub 2$\alpha$／were administrated, followed by administration of 5mg PGF／sub 2$\alpha$／ at the interval of 14 days. As results, 74％ (17／23 cows) of pregnancy rate after AI. In order to further reduce the open period, 5 mg PGF／sub 2$\alpha$／was administrated at the interval of 11 days without the period of uterus recovery, resulted in 94％ (16／17 cows) pregnancy rate. In conclusion, these results showed that PGF／sub 2$\alpha$／ and GnRH treatment were effective hormonal treatment resume in Hanwoo with various reproductive disorders. In addition, modified protocol of intraovarian PGF／sub 2$\alpha$／ administration could be the effective method for reducing the open period.

• Journal of Embryo Transfer
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• v.17 no.2
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• pp.145-151
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• 2002

The aim of these experiments was to investigate in vitro nuclear maturation of canine oocyte collected from various stages of estrus cycles, Ovaries were obtained from 1 to 4 year-old mongrel bitch and minced for oocyte collection in phosphate buffered saline with 100 iu penicillin-G $m\ell$／sup -1／, 50 $\mu\textrm{g}$ streptomycin sulphate $m\ell$／sup -1／ and 0.1％ (w／v) polyvinyl alcohol. Cumulus-oocyte-complexes (COCs) were washed in HEPES buffered tissue culture medium (TCM)199 and in vitro matured in TCM-199 culture medium supplemented with sodium pyruvate 0.028mg/$m\ell$, L-glutamine 0.146mg／$m\ell$, penicillin G 10,000 IU／$m\ell$, streptomycin 0.031mg／$m\ell$ and 10％ (v／v) fatal calf serum. COCs were in vitro matured for 48～72 hrs at 39$^{\circ}C$ in humidified 5％ $CO_2$ in air atmosphere. In vitro matured oocytes were remove the cumulus cells using 0.2％ (v／v) hyaluronidase. After denuding, oocyte were placed in acetic acid : methanol : chlorform solusion (3:6 : 1.5 v／v) for 30 sec and acetic acid: ethanol(1:3 v／v) for 48hrs fixation. Nuclear maturation was classified to GV, GVBD, MI, MII and degenerate oocyte under microscopy after 1％ aceto-orcein stain. In vitro maturation rates at 48hrs were not significantly difference among the oocytes collected from different stage of estrus at 15.9％, 16.3％, 23.7％ and 18.2％ for anestrus, proestrus, estrus and diestrus. However, the oocytes maturation(36.6％) of collected from estrus ovaries were significantly different from oocytes derived from proesturs, diestrus and anestrus ovaries(30.8％, 17.5％ and 22.1％; p＜0.05). The overall in vitro maturation rates were significantly higher (p＜0.05) in 72hrs culture than 48hrs culture system. In summary, there was a tendency for higher in vitro maturation rates with the oocyte collected from estrus ovary than other stages of estrus. Also, for nuclear maturation, in vitro culture of oocyte for 72hrs was better than 48hrs culture.

• Journal of Embryo Transfer
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• v.17 no.2
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• pp.137-143
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• 2002

This study was conducted to evaluate the effect of sugar kinds and combination of various sugars, and straw size and pre-freezing time on motility of post-thaw spermatozoa in canine. In general, the extender was supplemented with fructose or glucose for semen freezing. The extender used in .this study was Tris-citric acid extender (Tris-buffer) supplemented with 20％ Egg-yolk, 8％ glycerol, 1％ Equex STM paste, and 70 mM sugars such as monosaccharide (fructose and xylose) and disaccharide(trehalose). To evaluate of sugar combination, the sugars supplemented in Tris-buffer. were combined such as control (fructose, xylose, trehalose), two combinations (Fru＋Tre, Fru＋Xy1, Tre＋Xy1) and three combinations (Fru＋Tre＋Xy1). The motility after CASA analysis in Fru＋Tre＋Xy1 was higher than those in fructose, trehalose, xylose, Fru＋Tre, Fru＋Xy1, Tre＋Xy1 (69 vs. 58, 61, 50, 65, 20, 54). However, the progressive motility after CASA analysis in Fru＋Tre group was higher than those in fructose, trehalose, xylose, Fru＋Xy1, Tre＋Xy1, Fru＋Tre＋Xy1 (59％ vs. 47, 55, 42, 13, 49, 44％). The survival rate of post-thaw spermatozoa in 0.25 $m\ell$ straw for 10 min pre-freezing was significantly higher than those in 0.25 $m\ell$ straw for 10 min, 0.25 and 0.5 $m\ell$ for 5 min (80＋0.0 vs. 65＋7, 68＋16, 58＋8％; P＜0.05). The results indicated that the progressive motility of post-thaw spermatozoa in 70 mM Fru ＋Tre (two combination) following CASA analysis and 0.25 ml straw for 10 min pre-freezing time could be better for freezing of semen in canine.

• Journal of Embryo Transfer
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• v.17 no.1
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• pp.45-53
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• 2002

The main goal of this study was to produce transgenic porcine embryos by direct injection of sperm-mediated exogenous DNA. Spermatozoa (6$\times$10$^{6}$ sperms of final concentration) were mixed with pcDNA LAC Z (20 ng/$\mu$l) and subjected into electroporation (300~750 volts, 25 $\mu$F, 0.4 cm electrode). After sperm injection, the oocytes were activated electrically (1.7 KV/cm, 30$\mu$sec, single pulse) in 0.3 M mannitol solution or not. The sperm injected eggs were cultured in NCSU 23 medium (0.4% BSA) at 39$^{\circ}C$, 5% $CO_2$ in air fur 144 h. The rates of cleavage and development into blastocyst stage in activation group were significantly higher than those of non-activation group (79.6% and 24.1% vs. 46.3% and 14.4%, respectively, p<0.05). Control oocytes and shame injection were developed to blastocysts low (2.5%). Sixty five (27.1%) out of 240 embryos observed in activation and non-activation groups were showed positive by X-gal staining. However, all embryos in both groups were expressed partial or mosaic pattern. These results suggested that electrical stimulation far oocytes activation after sperm injection enhances the incidence of both fertilization and development fellowing sperm injection in the pig. Our study also suggested that sperm-mediated transfer of exogenous DNA by ICSI would be used as a valuable tool for the production of transgenic porcine embryos.

• Journal of Embryo Transfer
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• v.21 no.4
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• pp.315-322
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• 2006

This study was performed to investigate the physiological effects of bovine follicular fluid (bFF) or anti-inhibin serum (AI) on follicular development in Hanwoo. Saline (0.95%), bFF or AI (total of 40 ml) were administered into the jugular vein in 9 Hanwoo cows. The plasma inhibin, estradiol-17 $\beta$ (E2), and progesterone (P4) levels were measured using RIA or ELISA kit and the number of ovarian follicles was observed by ultrasonography at 72 hr after ovulation. The plasma inhibin level in bFF treatment group was significantly increased and maintained higher level from 102 hr after ovulation compared to that of saline and AI groups (p<0.05). In plasma E2 level, AI treatment group showed significantly higher level from 36 hr to 108 hr after ovulation than that of saline and bFF groups (p<0.05). After that it showed decreasing tendency. The plasma P4 level was increased in control and AI treatment groups at 68 hr after ovulation. However, it was maintained significantly lower level in bFF group from 84 hr to 180 hr compared to that of saline and AI group (p<0.05). As a result of ultrasonography at 72 hr after ovulation, higher number of follicles was shown in AI treatment group compared to bFF groups, although the difference was not statistically significant. Taken together, it can be postulated that a treatment of synthesized AI inhibits the secretion of inhibin, stimulates FSH secretion inhibited by inhibin, and induces follicular development and estrogen secretion. According to these results, a development of ovarian follicle immediately after ovulation is associated closely with inhibin in Hanwoo heifers.

• Journal of Embryo Transfer
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• v.21 no.4
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• pp.281-286
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• 2006

This study was conducted to investigate the hormonal changes in cultured medium during in vitro culture of bovine oviduct epithelial cells (BOEC) supplemented with interleukin (IL)-4 of 0.001, 0.01, 0.1 or 1 ng/ml. BOEC were collected from the oviduct and washed 3 times with 1% antibiotic-mycotic-DMEM medium and cultured at $39^{\circ}C$, 5% $CO_2$, 95% air for 24$\sim$120 hrs. The cultured media were analyzed hormonal changes with hormonal analyzing kit (progesterone (P4), estradiol (E2) : Perkin Elmer, USA) and Transforming growth factor (TGF)-$\beta$ with Eliza kit (Promega, USA). The production of P4 in 0.001 IL-4 was increased as the culture time increased. P4 production was significantly higher in the medium cultured for 120 hrs than 24 hrs (P<0.05). P4 production in 0.01 ng/ml group was similar to that of 0.001 ng/ml. The production of E2 in 0.001 and 0.01 ng/ml groups were increased to 72 hrs like P4 production and showed significantly different between the culture periods (P<0.05). After the culture for 96 hrs, P4 and E2 production were increased to 96 hrs, but decreased at 120 hrs. The production of TGF-$\beta$ showed no changes according to culture period or supplementation of IL-4. In conclusion, the supplementation of IL-4 can increase the production of P4 and E2 and might have important role for the successful pregnancy in bovine.

• Journal of Embryo Transfer
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• v.21 no.4
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• pp.331-338
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• 2006

This survey was carried out to investigate the occurrence of reproductive disorders in Korean native cattle (Hanwoo). The general management status such as barn type, feed intake, parity and type of reproductive disorders were examined in the Hanwoo farms. In this survey, the total incidence of reproductive disorders that was assessed from 54 Hanwoo farms was 11.6% (73/ 631). We first examined the incidence of reproductive disorders following herd size per farm. The rates of reproductive disorders in under 10 heads, 10 to 20 heads and over 20 heads were 25.2%, 9.4% and 8.3%, respectively (P<0.05). The rate of reproductive disorders in tie stall barn was significantly (P<0.05) higher than that in free stall barn (14.9% vs. 8.1%). The major types of reproductive disorders were follicular cysts (21.8%), fat necrosis (21.8%), and repeat breeding (17.8%). The rate of reproductive disorders assessed by the parity tended to decrease as the parity increased. The relationship between incidence of reproductive disorders and body condition score (BCS) was investigated in 203 Hanwoo. The incidence of reproductive disorder was 80.0%, 8.8%, 9.4%, 10.0%, 33.3% and 57.1% at 1.5, 2.0, 2.5, 3.0, 3.5 and 4.0 of BCS, respectively. These data show that the increase of herd size, barn type and BCS affect the incidence of reproductive disorders such as follicular cysts, fat necrosis and repeat breeding in Hanwoo.

• Journal of Embryo Transfer
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• v.19 no.3
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• pp.201-208
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• 2004

This study was performed to examine the effects of catalase and $\beta$-mercaptoethanol ($\beta$ME) on the establishment of clonal lines from porcine fetal fibroblast cells. Fibroblasts derived from a pig fetus (Day 50) were passaged two times before use. A single cell was seeded in 96-well plates and cultured in medium supplemented with or without catalase or $\beta$ ME. Cell colonies were passaged two times into 4-well dish. Cell lines with proliferating potential were classified as an established clonal cell line. In experience 1, the establishment efficiencies were examined by addition of catalase (100ng/$m\ell$) or $\beta$ME (100 uM) in culture medium. The establishment efficiency of $\beta$ME-added group (8.3％) was significantly higher than that of control group (3.2％, P<0.05). However, catalase did not have a positive efffct on the establishment efficiency. In experience 2, the establishment efficiencies were examined by addition of different concentrations of catalase (0-1,000 ng/$m\ell$) in culture medium. However, establishment efficiencies were not different among the different concentrations of catalase (0-2.6％). In experience 3. the establishment efficiencies were examined by addition of different concentrations of $\beta$ME(0-1,000 uM) in culture medium. The establishment efficiency was significantly higher in 100 uM $\beta$ME-added group (9.4％) compare to others (0-1.6％). The result of present study shows that the establishment efficiency of clonal cell lines can be enhanced by the culture in media supplemented with 100uM $\beta$ME. However, catalase did not have a positive effect on the establishment efficiency.

• Journal of Embryo Transfer
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• v.19 no.3
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• pp.219-227
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• 2004

This study was conducted to develop a serum-free, defined medium of IVM of pig oocytes. The TCM-199 with supplemented with polyvinylalcohol(PVA), polyvinylpyrrollidone(PVP) and porcine follicular fluid(pFF) were used as basal medium. The effects of the these additives on the rates of maturity and development under in-vitro fertilization and in vitro culture were examined and subsequently considered on the possibilities be sustituted for the bovine serum albumin(BSA). Maturation rate of pig oocytes in IVM media containing PVA(82.4％), pFF(89.4％) and BSA(90.0％) were significantly higher(P<0.05) than that of PVP(78.6％). Cleavage rate after IVF of PVP(64％) was significantly lower(P<0.05) than these of PVA(73％), pFF(77％) and BSA(73％) supplements. in vitro development rates to morulae and blastocyst on PVP(54％) were also significantly lower(P<0.05) than these of the supplements of PVA(63％), pFF(69％) and BSA(65％). In comparison of maturation and fertilization rates of pig oocytes in each supplements, the maturity rates of PVA(82.4％), pFF(89.4％) and BSA(90.0％) were significantly lower(P<0.05) than that of PVP(72.4％) and while, the fertilization rates of pFF(87.1％) and BSA(89.1％) were significantly higher(P<0.05) than these of PVA(78.0％) and PVP(70.6％). It may be concluded that PVA and pFF can be substituted far BSA in medium for culturing pig oocytes; however, it may be considered that PVP were limited to for BSA in the in vitro culture of the embryos.

• Journal of Embryo Transfer
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• v.22 no.1
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• pp.9-13
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• 2007

The objective of this study was to investigate the relationship between various estrous behavior and ovulation time, and to determine which estrous behavior could predict ovulation time more accurately. In total, 37 ovulations and 28 estrous detection were observed in 51 Holstein-Friesian dairy cows. Various estrous behavior were observed during 72 h from two days after $PGF_2{\alpha}$ injection and their relation with the time of ovulation (ultrasound examinations at 4-h intervals) was investigated. In estrous periods, the rate of sniffing, chin resting, mounting, standing heat was 81%, 78%, 78% and 56%, respectively. Ovulation occurred $25.6{\pm}7.9h$ after onset of estrus (ranging between 7 and 37h) and $13.4{\pm}7.1h$ after end of estrus (ranging between 1 and 28h). Interval between onset of estrus and ovulation time was significantly (p<0.05) shorter for standing heat $(17.33{\pm}5.83h)$ than for mounting, sniffing and chin resting $(23.58{\pm}5.12h,\;24.25{\pm}6.09h,\;23.42{\pm}6.04h)$. In 88% of the animals that displayed mounting, ovulation occurred between $16{\sim}28h$ after onset of mounting. Onset of standing heat, sniffing and chin resting occurred between $10{\sim}22(81%)h,\;16{\sim}28(79%)h\;and\; 19{\sim}31(79%)h$ before ovulation respectively. Sniffing and chin resting were displayed during the non-estrous period and are therefore, not useful predictors of ovulation time. The standing heat and mounting can be a good predictor for time of ovulation but the disadvantage of using standing heat is that only a limited number of cows display standing heat. Thus, it is concluded that mounting behavior could be the best predictor for time of ovulation.