• Journal of Embryo Transfer
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• v.21 no.1
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• pp.59-67
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• 2006

To evaluate the functional status of ovarian cyst in Korean native cows, progesterone ($P_4$) and estrogen ($E_2$) level of cystic follicular fluid, ultrasonography for measuring the cystic diameter and thickness of cystic wall, and histological findings were investigated in cystic ovaries from slaughtered Korean native cows. Ovarian cysts were classified as single follicular cyst 51 cows (59.3%), multiple follicular cysts 19 cows (22.1%), single luteal cyst 13 cows (15.1%) and multiple luteal cysts 3 cows (3.5 %) by anatomical and ultrasonography. Ovarian cysts were classified as follicular cysts (54 cows), luteal cyst (16 cows) and non-functional ovarian cyst (16 cows) by hormone analysis, anatomical finding and ultasonography The luteal cyst was accurately diagnosed by cystic wall thickness, but follicular cysts was misdiagnosed 16 cows of 70 cystic cows The cystic fluid $P_4$ concentration was 3.3 ng/ml in follicular cysts and 30.1 ng/ml luteal cysts. There was significantly positive correlations between cystic wall thickness and serum $P_4$ concentration in follicular ($r^2=0.59$, p<0.001) and luteal cysts ($r^2=0.65$, p<0.001). These results indicated that ovarian cysts had various stages of degeneration and luteal cyst was accurately diagnosed measurement of cystic wall thickness by ultrasonography, but follicular cysts were not diagnosed only cystic diameter and cystic wall thickness.

• Journal of Embryo Transfer
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• v.21 no.1
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• pp.21-27
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• 2006

This study was conducted to determine the distribution of cat follicles among varying ages and produce oocytes from preantral follicles cultured in vitro. We used ovaries from 41 cats ranging in age from 0.3 to 5 years. Ovaries were obtained from cats undergoing routine ovariectomy at local veterinary clinics. As a prelude to in vitro culture of preantral follicles, the length and the width and the weight of ovaries among cats of varying ages were measured. Ovaries were fixed in 10% formalin, embedded in paraffin, cut into $3{\mu}m$-sections, mounted on slides and stained with hematoxylin and eosin. Follicles were evaluated at 200X and 400X magnification. Distribution of follicles among cats of varying ages were evaluated according to follicle classification: primordial, primary, transitional, preantral and antral follicles. Preantral follicles were isolated by the simple mechanical procedure. Each follicle was cultured in a well containing $100{\mu}l$ of medium 199 supplemented with 10% fetal bovine serum (FBS) or polyvinylalcohol (PVA) for 16 days. Follicle diameters were measured under inverted microscope every 4 days. The length, the width and the weight of ovaries were increased gradually according to ages but there was not significant difference among cats of varying ages. Majority of follicles were primordial follicles (84%) regardless of cat ages (p<0.05). Follicle diameter increased until 4 days of culture. However, period longer than 4 days of culture in vitro had a deleterious effect on follicle survival regardless of supplement (FBS or PVA). A few oocytes were collected from preantral follicles cultured in vitro. These basic reproductive techniques in domestic cats can be a useful tool to save endangered feline species.

• Journal of Embryo Transfer
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• v.21 no.1
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• pp.35-43
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• 2006

Ions play important roles in various cellular processes including fertilization and differentiation. However, it is little known whether how ions are regulated during early embryonic development in mammalian animals. In this study, we examined changes in $Ca^{2+}\;and\;K^+$ concentrations in embryos and oviduct during mouse early embryonic development using patch clamp technique and confocal laser scanning microscopy. The intracellular calcium concentration in each stage embryos did not markedly change. At 56h afier hCG injection when 8-cell embryos could be Isolated from oviduct, $K^+$ concentration in oviduct increased by 26% compared with that at 14h after injection of hCG During early embryonic development, membrane potential was depolarized (from -38 mV to -16 mV), and $Ca^{2+}$ currents decreased, indicating that some $K^+$ channel might control membrane potential in oocytes. To record the changes in membrane potential induced by influx of $Ca^{2+}$ in mouse oocytes, we applied 5 mM $Ca^{2+}$ to the bath solution. The membrane potential transiently hyperpolarized and then recovered. In order to classify $K^+$ channels that cause hyperpolarization, we first applied TEA and apamin, general $K^+$ channel blockers, to the bath solution. Interestingly, the hyperpolarization of membrane potential still appeared in oocytes pretreated with TEA and apamin. This result suggest that the $K^+$ channel that induces hyperpolarization could belong to another $K^+$ channel such as two-pore domain $K^+(K_{2P})$channel that a.e insensitive to TEA and apamin. From these results, we suggest that the changes in $Ca^{2+}\;and\;K^+$ concentrations play a critical role in cell proliferation, differentiation and reproduction as well as early embryonic development, and $K_{2P}$ channels could be involved in regulation of membrane potential in ovulated oocytes.

• Journal of Embryo Transfer
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• v.21 no.1
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• pp.45-51
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• 2006

This study was carried out to investigate effect of claw trimming on milk yield and its composition in Holstein at different lactation stages. 1 . There was no difference in daily milk yield between control and claw trimming in early, mid and late lactating Holsteins. 2. Somatic cell count (SCC) was lower in early lactation and it was higher in late lactation when claws were trimmed in Holstein. However, claw trimming did not affect SCC during mid lactation in Holstein. 3. Milk fat, protein and total solids were decreased during late lactation in Holstein after claw trimming. However, milk composition was not affected by claw trimming in early and mid lactating Holsteins.

• Journal of Embryo Transfer
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• v.20 no.2
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• pp.123-128
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• 2005

This study examines the effects of kinds and concentrations of cryoprotectants on the survival rate of vitrification-thawed porcine oocytes, together with the effects on survival, in vitro fertilization and development of immature oocytes. 1. The developmental rate of oocytes to MII and diploid stage when the vitrification-thawed of recovered immature oocytes cultured for 0, 15, 30 and 40h were cultured for 0, 15, 30 and 40h were $56.7\%,\;53.3\%,\;63.3\%,\;65.0\%\;and\;23.3\%,\;18.3\%,\;10.0\%,\; 3.3\%$, respectively. The in vitro development to MII stage were lower than the control group $(78.2\%)$, but higher fo. diploid stage $(5.5\%)$. 2. When the vitrification of immature oocytes after being culture for 0, 15, 30 and 40 hours, the survival rate were $34.0\%,\;26.0\%,\;18.0\%\;and\;10.0\%$ respectively. This result was lower than that of the control group $(60.0\%)$. 3. When the fertilization of the vitrified immature oocytes after being culture for 0, 15, 30 and 40 hours, the in vitro fertilization rate were $60.0\%,\;54.0\%,\;48.0\%,\;38.0\%$, and developmental rates were $26.0\%,\;18.0\%,\;8.0\%,\;4.0\%$, respectively. This results were lower than the control group $(78.0\%\;and\;38.0\%)$. 4. When the fertilization of the immature oocytes after being culture for $0\~15$ hours vitrified with EDS and ETS, the fertilization and developmental rates were $50.0\%,\; 22.0\%$ and $46.0\%,\;18.0\%$, respectively. This results were lower than the control group $(74.0\%\;and\;38.0\%)$.

• Journal of Embryo Transfer
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• v.20 no.2
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• pp.129-135
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• 2005

The present study examines the effects of kinds and concentrations of cryoprotectants, PVP and sucrose and trehalose on the survival rate of vitrified porcine embryos. 1. The developmental stages for the embryos used in vitrification were $245(23.0\%)$ for 2 cell stage, $256(24.1\%)$ for the blastocyst, $234(22.0\%)$ for the early blastocyst $221(20.8\%)$ from the expanded blastocyst and $107(10.1\%)$ from hatching blastocyst out of 1,063 embryos. 2 The survival rate of morula, early blastocyst and expanded blastocyst vitrification-thawed with EDT and EGS were $69.1\%,\;70.3\%,\;69.8\%\;and\;62.5\%,\;61.7\%,\;63.6\%$, respectively. The expanded blastocyst treated with EDS showed the highest survival rate compared with the other cryoprotectants. 3. The survival rate of early blastocyst, expanded blastocyst and hatching blastocyst vitrification-thawed with EDS diluted in $medium + 10\%$ FCS were $61.1\%,\;27.8\%,\;16.7\%$, respectively. This result were love. than the control of group $(92.3\%,\;71.2\%,\; 55.8\%)$. 4. The survival rate of embryos vitrified with EDS and EDT supplemented with $10\%\;and\;20\%$ PVP were $74.3\%,\;77.5\%\;and\;79.4\%,\;71.1\%$, respectively. The survival rate of vitrified embryos cultured for $24\~48$ hours were $37.1\%,\; 40.0\%\;and\;35.3\%,\;31.6\%$ which were significantly lower than that of non-cultured embryos. The survival rate of embryos vitrified with EDS and EDT supplemented between $10\%\;or\;20\%$ PVP did not have a significant difference. 5. The survival rate of embryos vitrification-thawed with EDS to morula, early blastocyst, expanded blastocyst and hatching blastocyst were $58.2\%,\; 36.4\%,\;14.5\%$ to morula, $62.5\%,\;45.8\%,\;20.8\%$ to early blastocyst, $74.1\%,\;61.1\%,\;29.6\%$ to expanded blastocyst and $60.0\%,\;40.0\%,\;14.0\%$ to hatching blastocyst.

• Journal of Embryo Transfer
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• v.21 no.3
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• pp.255-262
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• 2006

The purpose of this study is to investigate the effects of vitrification in open pulled straws (OPS) methods on in vitro survival ability of porcine embryos. For in vitro maturation of immature oocytes, the porcine ovaries were collected from local slaughter-house. The cumulus-oocytes complexes were aspirated from 2 to 6 mm follicles. The collected oocytes were cultured for in vitro maturation in NCSU-23 medium with 5 mM hypotaurine, 0.57 mM cysteine, 10% porcine follicle fluid, 10 IU/ml PMSG and 10 IU/ml hCG for $21{\sim}22$ hrs. Then, the oocytes were more cultured $21{\sim}22$ hrs in vitro maturation in medium removed hormones. The frozen-thawed spermatozoa were washed by centrifugation 2 times for 10 min at 1,500 rpm in D-PBS with 5.56 mM glucose, 0.33 mM Na-pyruvate, 100 IU/ml penicillin, $100 {\mu}g/ml$ streptomycin and 4 mg/ml BSA. The fertilization medium used mTBM with 2 mM caffeine and 2 mg/ml BSA and adjusted to a pH of 7.2 to 7.4. The final concentration of spermatozoa was adjusted to $2.5{\times}10^6$cells/ml motile sperm during fertilization in vitro. At 8 hrs after insemination, the oocytes were transferred into NCSU-23 medium with 5.0 mM hypotaurine, 4 mg/ml BSA and 10 ng/ml EGF and cultured for 7 days. When the blastocysts of different stages were frozen-thawed by OPS methods, the proportions of embryos with normal morphology were significantly (p<0.05) higher in embryos frozen-thawed at expanded blastocyst stage (38.9%) than in early blastocyst stage (28.3%). On the other hand, the proportions of embryos damaged after frozen-thawing were significantly (p<0.05) higher in embryos frozen at early blastocyst stages than in expanded blastocyst stage. In another experiment, the normal embryos morphology after frozen- thawing were further cultured for 48 hrs. After culture, the proportions of embryos hatched were 6.7, 20.0 and 33.3% for embryos frozen-thawed at early blastocyst, mid-blastocyst and expanded blastocyst stages. These finding indicate the possible broader application for OPS methods, as frozen-thawed embryos may be accompanied by developmental stage according to requirements of the survival ability after freezing of blastocyst stage in the pig.

• Journal of Embryo Transfer
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• v.21 no.3
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• pp.177-182
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• 2006

This study was conducted to investigate the effects of melengesterol acetate (MGA) and $PGF_{2{\alpha}}$ administrations on serum progesterone level, synchrony of estrus and conception rates in Han-woo. Firstly, ten heifers and one freematin were fed 0.5 mg MGA/day for 14 days in a grain carrier, and after 19 days of MGA feeding, a single injection of 25 mg $PGF_{2{\alpha}}$ were treated. Blood samples were collected to evaluate serum progesterone concentrations from the start of feeding of MGA until the end of feeding and subsequent estrous detection and artificial insemination (AI) at 3 days intervals, and on days of $PGF_{2{\alpha}}$ injection, estrous detection, AI, and 15th and 60th days after AI. The level of progesterone in the blood began to increase from 7 days after MGA feeding, and 9 days after feeding it became 5.4 ng/ml and maintained that level thereafter. On the 33th day when the $PGF_{2{\alpha}}$ was injected, it reached the peak level of 7.6 ng/ml. However, 2-3 days after $PGF_{2{\alpha}}$ injection, it dropped to 1.4 ng/ml drastically (p<0.05). Secondly, one hundred and ninety four Hanwoo heifers or cows were divided into two groups to compare estrous induction and conception rates: the one treated with MGA and $PGF_{2{\alpha}}$, (n=104) and the other with $PGF_{2{\alpha}}$ treatment (two injections at 11 days interval, n=90). The heifers or cows treated with MGA and $PGF_{2{\alpha}}$ were identical to those used as above. The percentages of heifers or cows showed estrus were higher in the $MGA+PGF_{2{\alpha}}$ treatment (91.3%) than in the $PGF_{2{\alpha}}$ treatment (72.2%, p<0.05). Conception rates were also higher in the $MGA+PGF_{2{\alpha}}$ treatment (94.2%) than in the $PGF_{2{\alpha}}$ treatment (88.9%, p<0.05). The results of this experiment indicate that estrus synchronization using $MGA+PGF_{2{\alpha}}$ is more effective than that using $PGF_{2{\alpha}}$ (two injections) in Hanwoo.

• Journal of Embryo Transfer
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• v.21 no.3
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• pp.263-271
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• 2006

The purpose of this study was undertaken to compare ability of frozen-thawed sperm characteristics between two strains (miniature pig and Duroc). The semen was collected by gloved-hand method into a pre-warmed ($37^{\circ}C$) thermos bottle. The semen was diluted with same volume extender and added to LEY solution for freezing. The diluted semen was placed in 0.5 ml straws, and freezing was initiated by exposing the straws to liquid nitrogen ($LN_2$) vapours for 10 min before placing them into $LN_2$ for cryopreservation. The frozen-semen straw were thawed at 20, 37 and $50^{\circ}C$ for 1 min, 45 sec and 10 sec within water-bath. The semen sample were evaluated at 0, 3, 6, 9, and 12 h after incubation at $37^{\circ}C$ for analysis of sperm ability. Abnormality of spermatozoa in miniature pig was significantly (p<0.05) higher than that in Duroc at 0, 9 and 12 h of post-thawing incubation after frozen-thawing. The percentage of F-patterned spermatozoa in miniature pig was significantly (p<0.05) lower, while the percentage of AR (acrosome reacted spermatozoa) pattern was higher in the miniature than in the Duroc. On the other hand, there was no significant difference in the viability of spermatozoa thawed at different temperature ($20^{\circ}C\;and\;37^{\circ}C$) between two species, but the viability in miniature pig was higher (p<0.05) than in Duroc when sperm was thawed at $50^{\circ}C$. In conclusion, this study suggest that suitable freezing method for miniature pig semen is required for increasing post-thawing viability and fertilization capacity.

• Journal of Embryo Transfer
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• v.21 no.3
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• pp.207-216
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• 2006

Vaginal cytology was examined in 12 Shih-tzu bitches to establish the accurate basic data for estimate to the optimal mating time and ovulation time. The mean duration of proestrus and estrus were $9.09{\pm}0.83\;(mean{\pm}SD)$ days and $7.36{\pm}0.47$ days in pregnant bitches. The gestational length in the 12 pregnant bitches was $65.2{\pm}0.5$ days in pregnant bitches when Day 0 was timed from the first day of male acceptance. Characteristic features of vaginal cytology during the estrous cycle were the high proportion of large intermediate cell, superficial cell, anuclear cell and erythrocyte in proestrus, superficial cell, anuclear cell and erythrocyte in estrus, and parabasal cell, small and large intermediate cell and leukocyte in diestrus, respectively. Cornification index (CI) was the high proportion in proestrus and estrus, then it decreased in diestrus and anestrus. When Day 0 was timed from the day of the first male acceptance, the CI peak was Day 2 and maintained above 80% between Day -4 and Day 6 during 11 days, and above 90% between Day -1 and Day 5 during 7 days. In relationship between CI and reproductive hormones, CI showed peak at the first day after plasma estradiol-$17{\beta}$ concentration peak and plasma progesterone concentration was first increased above 4.0 ng/ml at Day 0 which was the first day after CI peak. In conclusion, ovulation in Shih-tzu bitches occurred at the first day after CI peak. Vaginal cytology is the simple and reliable method for estimating estrous cycle, optimal breeding time and ovulation time in Shih-tzu bitches.