• 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.

• Journal of Embryo Transfer
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• v.22 no.1
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• pp.63-67
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• 2007

This study was performed to evaluate the efficacy of sodium carboxymethylcellulose (SCMC) for the prevention of postoperative uterus and ovary adhesion formation in the Korean black goats. Twenty adult female Korean black goats randomly were divided into five groups with four animals in each group. After the routine laparotomy, catheter for injection was inserted into the abdomen. Before abdominal closure, saline, 1% SCMC, 2% SCMC or 0.4% HA solution (50ml/10kg of body weight/head) were injected in the abdominal cavity in each group. Three weeks after surgery, second laparotomy was performed and the adhesions were scored on a scale of 0 to 10 according to their vascularity and adhesion size in uterus and ovary. This trial was repeated the four times with the interval of three weeks. In the first and second trial, the group treated with 2% SCMC significantly reduced the adhesion formation than other treatment groups (P<0.05). In the third trial, the adhesion formation was significantly reduced in 2% SCMC and 1% SCMC (P<0.05). In the fourth trial, 2% SCMC reduced the adhesion formation. However, there was no significant difference among other groups. This study showed that the 2% SCMC administered at the end of the surgery reduced the adhesion formation in the Korean black goats.

• Journal of Embryo Transfer
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• v.22 no.1
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• pp.27-32
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• 2007

This study was conducted to examine the effects of prostaglandin $F_2{\alpha}(PGF_2{\alpha})$ and prostaglandin $E_2 (PGE_2)$ on the expansion and hatching of bovine embryos. During the in vitro culture, embryos were cultured with the following groups: (1) 0, 1, 10 and 100ng/ml $PGF_2{\alpha}$ (2) 0, 1, 10 and 100 ng/ml $PGF_2{\alpha}$, (3) low concentration of $PGF_2{\alpha}$ ; low concentration of $PGF_2{\alpha}$, (1ng/ml : 1ng/ml), (4) low concentration of $PGF_2{\alpha}$ : high concentration of $PGF_2{\alpha}$ (1ng/ml : 10ng/ml) (5) high concentration of $PGF_2{\alpha}$ : low concentration of $PGE_2$ (10ng/ml 1ng/ml) (6) high concentration of $PGF_2{\alpha}$ : high concentration of $PGE_2$(10 ng/ml : 10ng/ml). In the results of this study, treatment of $PGF_2{\alpha}$ or $PGE_2$ did not affect in vitro development to blastocysts. However, the hatching rates of embryos cultured with 10ng/ml $PGE_2$(10.3%) and 1ng/ml $PGF_2{\alpha}$ 10ng/ml $PGE_2$(22.2%) were significantly (P<0.05) higher than in control (4.3% and 12.7%) and other treatment groups. All groups treated with high concentrations of $PGF_2{\alpha}$ showed decreased hatching rates. Thus, this results suggested that $PGF_2{\alpha}\;and\;PGE_2$ were concerned with the hatching in bovine embryos, and their effects on hatching were different by the concentrations.

• Journal of Embryo Transfer
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• v.22 no.1
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• pp.15-19
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• 2007

The objective of this study was to investigate the relationship between body condition score (BCS), blood urea nitrogen (BUN) and estrous expression for the purpose of improving reproductive performance. In total, 37 ovulations and 28 estrous detection were observed among 51 Holstein-Friesian dairy cows. The estrous inducement rate and estrous expression rate were significantly lower for cows with BCS below 2.0 than for cows with BCS above 2.0. There was 0% of rate of standing heat in cows with BCS below 2.0 whereas the rate of standing heat was markedly increased in cows with BCS above 2.0 (46.7% and 64.7% for BCS $2.0{\sim}2.49$ and BCS $2.5{\sim}3.0$ cows, respectively). The estrous expression rate was significantly lower for cows with BUN below 10mg/dl than for cows with BUN above 10mg/dl. There was no significant difference among duration time of estrus, estrous behavior patterns and BUN concentration. The rate of estrous expression and concentration of BUN was not significantly different between primiparous and multiparous cows. This result shows that the level of BCS and BUN affect the estrous expression. Considering the situation that estrous expression is decreased in recent years, effective nutritional management should be accompanied to improve reproductive performance.

• Journal of Embryo Transfer
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• v.28 no.3
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• pp.177-184
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• 2013

The objective of this study was to analyze the accuracy of estrus detection of heat detector and analysis of estrus behavior (mounting and mounted), and the evaluation of conditions required for improving reproductive efficiency in Holstein dairy cows fitted with a estrous detector. The heat detection system consists of estrous detector based on wireless sensor and an electric bulletin board displayed estrus behavior data. When cow mounting other cows, the accuracy of estrus behavior displayed an electric bulletin board were 87.5% (mounting other cows only), 100% (mounting other cows but not standing), 80.0% (mounting other cows with standing for 1~4 seconds), 90.0% (mounting other cows but not standing for 1~4 seconds), 80% (mounting other cows with standing for more than 5 seconds) and 90.0% (mounting other cows but not standing for more than 5 seconds). When cow mounted other cows, the accuracy of estrus behavior displayed an electric bulletin board were 100% (mounted other cows but not standing), 100% (mounted other cows with standing for 1~4 seconds), 100% (mounted other cows but not standing for 1~4 seconds) and 100% (mounted other cows with standing for more than 5 seconds). Circadian distribution of first observed in estrus were 59.1% (am 8~pm 6) and 40.9% (pm 6~am 8). Distribution for the number of estrus behavior were 40.9% (less than 3 times), 36.4% (4~6 times) and 22.7% (more than 4 times). The conception rates relative to interval from first estrus behavior to insemination for estrus periods were 23.1% (less than 11 hours) and 55.6% (12~20 hours).

• Journal of Embryo Transfer
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• v.28 no.3
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• pp.281-287
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• 2013

A major barrier to progress in pig to primate organ transplantation or cell therapy is the presence of terminal ${\alpha}$-1,3-galactosyl epitopes on the surface of pig cells. Therefore, the purpose of this experiment was to establish and cha- racterize mesenchymal stromal/stem cells (MSCs) derived from ${\alpha}$-1,3-galactosyltransferase (GalT) knock out (GalT KO) pig to confirm their potential for cell therapy. Bone marrow (BM)-MSCs from GalT KO pig of 1 month old were isolated by Ficoll-Paque PLUS gradient and cultured with A-DMEM + 10% FBS on plastic dishes in 5% $CO_2$ incubator at 38.5. GalT KO BM-MSCs were analyzed for the expression of CD markers ($CD45^-$, $29^+$, $90^+$ and $105^+$) and in vitro differentiation ability (adiopogenesis and osteogenesis). Further, cell proliferation capacity and cell aging of GalT KO BM-MSCs were compared to Wild BM-MSCs by BrdU incorporation assay (Roche, Germany) using ELISA at intervals of two days for 7 days. Finally, the cell size was also evaluated in GalT KO and Wild BM-MSCs. Statistical analysis was performed by T-test (P<0.05). GalT KO BM-MSCs showed fibroblast-like cell morphology on plastic culture dish at passage 1 and exhibited $CD45^-$, $29^+$, $90^+$ and $105^+$ expression profile. Follow in ginduction in StemPro adipogenesis and osteogenesis media for 3 weeks, GalT KO BM-MSCs were differentiated into adipocytes, as demonstrated by Oilred Ostaining of lipid vacuoles and osteocytes, as confirmed by Alizarinred Sstaining of mineral dispositions, respectively. BrdU incorporation assay showed a significant decrease in cell proliferation capacity of GalT KO BM-MSCs compared to Wild BM-MSCs from 3 day, when they were seeded at $1{\times}10^3$ cells/well in 96-well plate. Passage 3 GalT KO and Wild BM-MSCs at 80% confluence in culture dish were allowed to form single cells to calculate cell size. The results showed that GalT KO BM-MSCs($15.0{\pm}0.4{\mu}m$) had a little larger cell size than Wild BM-MSCs ($13.5{\pm}0.3{\mu}m$). From the above findings, it is summarized that GalT KO BM-MSCs possessed similar biological properties with Wild BM-MSCs, but exhibited a weak cell proliferation ability and resistance to cell aging. Therefore, GalT KO BM-MSCs might form a good source for cell therapy after due consideration to low proliferation potency in vitro.

• Journal of Embryo Transfer
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• v.18 no.2
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• pp.91-96
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• 2003

The aim of this study was conducted to evaluate the effect of sugar kinds and combination of various sugars on acrosome damage of post-thaw spermatozoa in canine. 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+Xyl, Tre+Xyl) and three combinations (Fru+Tre+Xyl). The acrosome damage rate of post-thaw spermatozoa in Eosin B & Fast Green stain in Fruc+Tre was higher than those in fructose, trehalose, xylose, Fruc+Xyl, Tre+Xyl, Fruc+Tre+Xyl (83.0$\pm$5.6 vs. 82.3$\pm$3.1%, 81.7$\pm$2.1%, 72.0$\pm$2.0%; 80.3$\pm$4.5%, 76.7$\pm$3.8%, 81.0$\pm$5.6). The motility after CASA analysis in Fru+Tre was higher than those in Fru+Tre+Xyl, Tre+Xyl, Fru+Xyl, Xylose, Trehalose, Fructose(79$\pm$6 vs 75$\pm$3, 74$\pm$8, 71$\pm$11, 70$\pm$4, 66$\pm$15, 63$\pm$ 12%). However, the progressive motility after CASA analysis in Fru+Tre group was higher than those in Fru+Tre+Xyl, Tre+Xyl, Fru+Xyl, Xylose, Trehalose, Fructose (67$\pm$7, 64$\pm$3, 62$\pm$6, 61$\pm$8, 60$\pm$2, 57$\pm$13, 53$\pm$10%). The results indicated that the acrosome damage & progressive motility of post-thaw spermatozoa in 70 mM Fruc+Tre (two combination) following Eosin B & Fast Green stain and CASA analysis.

• Journal of Embryo Transfer
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• v.18 no.2
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• pp.115-124
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• 2003

The objective of this study was to compare the effect of four different estrus induction methods in anestrus cows on the estrus induction and pregnancy following artificial insemination (AI). Sixty-five cows (3∼4 years old) were selected and divided into four different estrus induction treatment groups. Group 1, 12 cows were treated by Ovsynch program combined with GnRH and PGF$_2$a. Group 2, 12 cows were treated by "Tow plus Two" program with GnRH and PGF$_2$a. Group 3, 20 cows were treated by "Tow plus Two" program following intravaginal progesterone implantation (CIDR). Twenty one cows in Group 4 were treated by "Tow plus Two" program following follicular rupture and intravaginal progesterone implantation. Cows were then observed estrus induction and inseminated artificially at 12 h and 24 h after standing estrus. The rates of estrus induction in Group 4 (18/21, 86%) was significantly (P<0.05) higher than those in groups 1, 2 and 3 (8/12, 67%; 9/12, 75%; 14/20, 70%). In the mean time of onset of estrus after final administration of GnRH in different hormone-treated cows, the cows in Group 3 (24.2$\pm$2.2) and Group 4 (23.4$\pm$2.0) were significantly (P<0.05) shorter than that in Group 1 (28.5$\pm$4.6) and Group 2 (26.4$\pm$3.3). The rates of pregnancy diagnosed on Day 28 were significantly different between treatment groups. Significantly (P<0.05) higher rate of pregnancy was observed in Group 4 (17/20, 85.0%) than those in Groups 1, 2 and 3 (7/11, 63.6%; 8/12, 66.7%; 15/20, 75.0%, respectetively). The rate of abortion diagnosed on 49 days of gestation was significantly (P<0/05) lower in Group 4 (1/17, 5.9%) than those in Groups 1, 2 and 3 (2/7, 28.7%; 2/8, 25% and 3/15, 20%, respectively). In conclusion, combined treatments with GnRH and PGF$_2$a following follicular rupture and progesterone implant in anestrus cows was considered to be most effective in estrus induction and maintenance of pregnancy. Further studies are needed to verify the functional mechanisms of residual follicles in anestrus ovaries on retarding the response of hormonal treatments.sponse of hormonal treatments.

• Journal of Embryo Transfer
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• v.16 no.2
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• pp.107-115
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• 2001

Development of effective activation protocols is of great importance for improving the success of cloning and subsequent transgenic. Three methods for oocyte activation, including 5$\mu$M ionomycin (5 min) alone, ionomycin + 1.9 mM 6-dimetylaminopurine (DMAP, 3 hrs) and ionomycin + 10 $\mu\textrm{g}$/ml cycloheximide (CHX, 3 hrs) were compared for their effects of pronuclei (PN) formation, development, developmental velocity and ploidy of parthenotes to IVF control in bovine. In group of ionomycin + DMAP, the oocytes having more 3PN were significantly (P<0.05) higher than in groups of ionomycin alone and of ionomycin + CHX (45.5% vs. 0 and 0%, respectively). Activation with the ionomycin alone, ionomycin + DMAP and ionomycin + CHX resulted in cleavage rates of 30, 85.5 and 57.9%, respectively. The blastocysts rate of parthenotes activated by ionomycin + DMAP treatment was significantly higher (12.3%. p<0.05) than those of other treated groups. Chromosome analysis shows that ionomycin + DMAP treatment greatly enhances the incidence of chromosomal abnormality of the parthenotes. From the results, we may conclude that DMAP treatment to the oocytes accelerates developmental velocity resulting in both the higher incidence of chromosome abnormality and of PN formation, and strongly suggest that CHX combined with ionomycin is better than DMAP for the purpose of successful nuclear transplantation. Developmental velocity of parthenotes activated by ionomycin + DMAP treatment was significantly (P<0.05) faster than others.