• Asian-Australasian Journal of Animal Sciences
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• v.19 no.2
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• pp.171-175
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• 2006

The aim of this in vitro study was to test the effect of microinjection (Mi) of foreign gene into the rabbit egg pronucleus and epidermal growth factor (EGF) addition on the blastocyst rate, the cell number and the diameter of embryos, and to determine possible relationships between embryo cell number and embryo diameter. Blastocyst rate was significantly decreased in gene- Mi (G-Mi/E0) group (63.1%) comparing to intact ones (83.5%, $p_1$<0.05). The addition of EGF at 20ng/ml (G-Mi/E20) or 200 ng/ml (GMi/ E200) to gene-Mi embryos did not affect blastocyst rate (65.6 and 55.2% resp.). As a control for Mi, the eggs were microinjected with the same volume of phosphate-buffered solution (PBS-Mi) instead of the gene construct solution. Cell numbers and embryo diameters were measured from embryo images obtained on confocal laser scanning microscope. Bonferroni-modified LSD test showed that the embryo cell number in PBS-Mi group was significantly lower ($p_1$<0.05) and in gene-Mi group was tended to decrease compared with intact embryos. Embryo diameter was not different among experimental groups. No effect of EGF given at any doses both on the cell number and embryo diameter was found. A positive correlation between cell number and embryo diameter was observed in all groups of embryos. Since embryo diameter was not changed under the influence of Mi or EGF addition in this study, this seems to be more conservative characteristics of the embryo morphology. These results suggest that the pronuclear microinjection compromises developmental potential of embryos, decreasing blastocyst rate and embryo cell number, whilst embryo diameter is not affected. No effects of EGF on studied parameters were confirmed. Declined quality of Mi-derived embryos is caused by the microinjection procedure itself, rather than by the gene construct used.

• Asian-Australasian Journal of Animal Sciences
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• v.11 no.3
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• pp.293-299
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• 1998

This study was undertaken to measure the concentrations of estradiol-$17{\beta}$, progesterone and testosterone, and to study their relationship with each other and with follicular size in individual buffalo ovarian follicles categorized as small (4 to 5 mm diameter), medium (6 to 9 mm diameter) and large (${\geq}10mm$ diameter). Steroid hormone concentrations varied markedly within follicles of each size category. Estradiol-$17{\beta}$ concentrations (pmol/ml) were positively related to follicular diameter (R = 0.34, n = 308, p < 0.001) and were significantly higher (p < 0.001) in large (1$118.46{\pm}30.25$), compared to those in medium follicles ($50.32{\pm}8.29$) which, in turn were significantly higher (p < 0.001) than those in small follicles ($19.70{\pm}$5.57). Progesterone and testosterone concentrations (pmol/ml) were not related to follicular diameter and were not different among small ($330.99{\pm}27.32$ and $17.68{\pm}2.44$ respectively), medium ($384.84{\pm}26.20$ and $36.47{\pm}4.55$, respectively) and large follicles ($253.25{\pm}32.23$ and $22.57{\pm}4.48$, respectively). Estradiol-$17{\beta}$ and progesterone concentrations were positively related (R = 0.39, n = 47, p < 0.01) in small, unrelated in medium and negatively related in large follicles (R = -0.59, n = 23, p < 0.01). There was no relationship between estradiol-$17{\beta}$ and testosterone concentrations in follicles of all the three size categories. Progesterone and testosterone concentrations were positively related in large follicles (R = 0.57, n = 18, p < 0.02). There was no relationship between the two hormones in small and medium sized follicles. When the follicles with estradiol-$17{\beta}$/progesterone molar ratios of > 1.00 were considered non-atretic, and the rest at different stages of atresia, 197/208(95%) follicles were found to be atretic.

• KOREAN JOURNAL OF CROP SCIENCE
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• v.50 no.5
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• pp.356-360
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• 2005

Mature embryos were aseptically excised with a scalpel and sliced in fragments measuring 0.5 mm in diameter (sliced mature embryo fragment; 4 ${\~}$ 5 fragments/one embryo). Sliced mature embryo fragments of six wheat cultivars were cultured to develop an efficient method of callus induction and plant regeneration. Callus derived from sliced mature embryo fragments showed a good capacity to embryogenesis and regeneration. Furthermore sliced mature embryo fragments decreased contamination from fungi and bacteria. The high efficiency of callus induction were obtained Keumkangmil and Bob­white. For plant regeneration, selected embryogenic calli were transferred to two types regeneration media. An average number of green spots per callus was 4 to 5 in regeneration media after about one week. Percentage of plant regeneration showed high in regeneration medium containing 0.1 mg/l 2,4-D and 5 mg/l zeatin. Especially, Keumkangmil ($27.5\%$) and Bobwhite ($33.3\%$) showed high regeneration efficiency. This regeneration system from sliced mature embryo fragments may provide an effective and convenient explant for plant transformation studies.

• Journal of Plant Biotechnology
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• v.42 no.3
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• pp.239-244
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• 2015

Calanthe striata f. sieboldii Decne. ex Regel is a terrestrial orchid with beautiful flowers arranged in racemose inflorescences. This species is threatened due to over-collection and loss of suitable habitats. Asymbiotic germination is useful in the conservation efforts to re-establish plants in the wild, and for commercial propagation. In this study, we investigate the effects of sodium hypochlorite (NaOCl), three types of culture media (Phytomax Orchid Maintenance - POM, Seed Germination Maintenance - SGM, and Murashige and Skoog 1962 - MS), and plant growth regulators on embryo swelling, protocorm formation, and embryo diameter of C. striata f. sieboldii. Treatment with 1% NaOCl for 30 min greatly enhanced embryo swelling (28.3%), embryo diameter ($205.8{\mu}m$), and embryo protocorm formation (54.8%) compared to seeds without NaOCl treatment (embryo swelling 8.5%, embryo diameter $14.6{\mu}m$, and protocorm formation 13.4%) on POM medium. Protocorm formation on POM medium supplemented with 1.0 mg/L N6-benzyladenine (BA) (95.6%) was better than the control (54.5%). Additionally, the effects of activated charcoal (AC) and sucrose on seedling growth in in vitro culture were examined. The protocorm converted into healthy plants with well-developed shoot primordia on the POM medium with AC and sucrose. The most suitable conditions for seedling growth after 10 weeks of culture were the POM medium with AC or sucrose. These results show effective asymbiotic seed germination and in vitro seedling growth of C. striata f. sieboldii.

• Journal of Embryo Transfer
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• v.8 no.1
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• pp.13-19
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• 1993

These experiments were carried out to determine the effect of pregnancy in bisected embryo. The embryos of ICR mouse were microsurgically bisected at morula and blastocyst stage using microsurgical blade attached a micromanipulator. These bisected embryos without zona pellucida were cultured up to blastocyst stage and cell count and diameter of stained blastomere, and transferred pseudopregnant mice. And the development of these bisected embryos was compared with the results of production of young of the corresponding intact embryos or cell stage. When the bisected mouse embryos were cultured in vitro for 20 to 24 hours in morula stage(77.2%) or 3 to 6 hours in blastocyst stage(84.1%), them were developed to the expanded blastocyst stage. There were no significant(P<0.05) differences in the development rate of bisected embryos between in morula and blastocyst stages. The embryo size of blastocyst developed in vitro from bisected embryo was small(P<0.05)than intact embryo. However, the number of blastomeres with bisected embryo (24.7+1.3and 21.5+1.2 respectively) were significantly(P<0.05) reduced, compared with that of intacted embryos(36.3+1.1 and 41.4+1.2 respectively). When compared with the result of pregnancy rate(63.6%) after surgical transfer of bisected morulae, a similar result(65.4%) was obtained with bisected blastocyst stage(P< 0.05). However, production of youngs (38.8%) after transfer of bisected morula, a similar result (38.1%) was obtained with bisected blastocyst stage (P<0.05).

• Journal of Embryo Transfer
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• v.29 no.4
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• pp.369-373
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• 2014

This study was performed to establish a new parameter for estimating gestational age and predicting parturition day by ultrasonographic measurement of deep portion of telencephalic vesicle (DPTV) diameter in small dogs. Fetal head diameter (HD) and DPTV diameter were measured in 15 pregnant Pekingese bitches, from Day 15 to the parturition day, and evaluated the correlation between gestational age. HD was measured from day 29 of pregnancy to parturition day and increased from $4.9{\pm}2mm$ to $25.5{\pm}0.7mm$. Especially, from day 38 of pregnancy to parturition day, HD uniformly increased about 0.6 mm per day and was significantly and linearly relative to gestational age during this period ($r^2$ >0.99). DPTV diameter was measured from day 35 to day 60 of pregnancy and increased from $3.2{\pm}0.9mm$ to $11.5{\pm}0.7mm$. Especially from day 38 to day 60 of pregnancy, DPTV diameter uniformly increased about 1 mm per 3 days and was significantly and linearly relative to gestational age during this period ($r^2$ >0.99). In conclusion, DPTV diameter could to be a useful parameter for the estimation of gestational age and the prediction of parturition day when used alone or in combination with HD during the second half of pregnancy.

• 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.

• Korean Journal of Animal Reproduction
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• v.19 no.3
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• pp.181-189
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• 1995

In this study, methods on fabrication of microtool and setting of micromanipulator were examined and relationship between first polar body extrusion rate and maturation time of follicular oocyte, enulceation rae and repetition of trial, and enucleation rate and maturation period were investigated. The results are as follows: 1. Suitable outside diameter of micropipette tube was 1mm. Holding pipette with less than diameter of oocyte was fitred for manipulation, and zona dissection needle was easily operated when its sharp-point had diameter of about 8 ${\mu}{\textrm}{m}$ and length of 300${\mu}{\textrm}{m}$. The injection pipette with 20~35${\mu}{\textrm}{m}$ outside diameter was adequate for injection of blastomere into perivitelline space. 2. Separation of blastomere was effective when zona pellucida had cut with zonadissection needle and the embryo was pipetted gently with the pipette that had narrower diameter than that of embryo until separation of blastomeres had completed. 3. The extrusion rate of first polar body was 78% during 20~24% hours incubation for maturation. 4. According to repetitions of micromanipulation, the enucleation rate was increased to 85% and the time required for enucleation of a oocyte was shortened to 3 min. 5. The extrusion rate of first polar body and enucleation rate were 82 and 76% respectively, in the group of the oocytes cultured for 22 hours. However in the group cultured for 24 hours, the extrusion rate of first polar body and enucleation rate were 53 and 100% respectively.

• Journal of Embryo Transfer
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• v.23 no.3
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• pp.147-153
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• 2008

Serial ultrasonographic examinations were daily performed from 15 days after ovulation until parturition to determine the growth curve of gestational structures in pregnant Maltese, Yorkshire terrier, Shih-tzu, and Miniature Schnauzer bitches, respectively. Gestational age was timed from the day of ovulation (day 0), which was estimated to occur when plasma progesterone concentration was first increased above 4.0 ng/ml. The inner chorionic cavity diameter were significantly and linearly relative to gestational age especially days 20 to 40, and the fetal head diameter were significantly and linearly relative to gestational age especial1y day 40 to parturition. These results indicate that inner chorionic cavity diameter were the most accurate for estimating gestational age before day 38 of gestation and the fetal head diameter were after day 38 of gestation.

• Journal of Embryo Transfer
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• v.22 no.4
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• pp.229-234
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• 2007

This study was undertaken to determine the normal appearance of the postpartum uterine involution. Postpartum changes in uterine shape, architecture, echogenicity and diameter were monitored with ultrasonography in 8 Miniature Schnauzer dogs. Vaginal discharge was observed visually during the 3 weeks of postpartum. A large amount of viscous dark green discharge changed progressively to a small amount of transparent discharge. In the uterine shape, the transverse images were crescent or polygonal at the beginning, but became circular after 16 days. At postpartum day 24, the longitudinal images of placental and interplacental sites were similar to each other The echogenicity of uterine structure was clearly distinguishable among the hyperechoic serous membrane, hypochoic myometrium, hyperechoic endometrium, and anechoic uterine contents during the first week postpartum, but afterward the interluminal boundaries and echogenicity became obscure gradually. In addition, the anechoic substance of the endometrial cavity was not observed after postpartum day 28. In the uterine diameter, the diameter of placental sites decreased markedly from 24 mm at the first day postpartum to 13 mm at day 7, and the diameter of interplacental sites decreased from 15 mm at postpartum day 1 to 10 mm at day 7. At postpartum day 65, the uterus (6.4 mm) changed little and had a uniform homogenous echo, and thus it was confirmed that uterine involution was completed at that time in Miniature Schnauzer dogs.