• Journal of Embryo Transfer
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• v.10 no.1
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• pp.45-51
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• 1995

In order to determine the optimum condition and timing for in vitro maturation of oocytes to metaphase of meiosis II (M II), the immatured follicular oocytes were recovered by puncturing the large(1.0~1.5 mm in diameter) and small(<1.0 mm in diameter) follicles in the ovaries of rabbits treated intramuscularly with a single dose of 100 TU PMSG 68 hours previously. The follicular oocytes were classified into three grades by the attachment of cumulus cells. The Grade I and II follicular oocytes from large follicles were cultured in BO-DM medium with 10% FCS, 35 $\mu$g /nl of FSH, 10 $\mu$g /ml of LH and 1 $\mu$g /ml of estradiol-17$\beta$ at 39t in a 5% $CO_2$ incubator for 11 to 23 hours. In 3 hours interval during the culture period, the oocytes were harvested and their cumulus cells were removed with hyaluronidase. The denuded oocytes were stained with Hoechst 33342 dye and their meiotic status and extrusion of the first polar body (PB) were examined under a fluorescence microscope. Also the fragmentation of the first PB and the distance between the first PB and nucleus were examined. The results obtained were as follows: 1. The mean recovery rate of follicular oocytes from the large and small follicles was 59. 9 and 31.3%, respectively. The mean number of oocytes recovered per rabbit and the Grade I percentage were 14.6 and 94.4% in large follicles, but 2.1 and 61.1% in small follicles, respectively. All the parameters examined were different significantly (p<0.05) between both the folliclular size. 2. Most of the follicular oocytes(86.8%) were matured in vitro to M II phase in 14 hours in Grade I oocytes, but the significantly(p<0.05) less oocytes(45.5%) were matured in Grade II oocytes. 3. The first PB was extruded in most of the oocytes(94.7%) in 14 hours of culture with the fragmentation rate of 29.6%, but the fragmentation rate of the first PB increased significantly (p<0.05) as the culture period for maturation was longer to 20 hours(63.5%). 4. The distance between the first PB and nucleus was increased linearly (p<0.05) as the maturation time passed from 14(7.1$\mu$rn) to 23 hours(58.4$\mu$m). 5. From the above results it was concluded that the optimum time for in vitro maturation culture might be 14 hours in the follicular oocytes from rabbit primed with PMSG for 68 hours, expecially when these follicular oocytes were used for recipient cytoplasms in embryo cloning.

• Journal of Embryo Transfer
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• v.11 no.3
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• pp.259-269
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• 1996

The present study was undertaken to examine the critical effect of $Ca^2$+ concentration on electrostimulation and post-electrostimulation media for electric activation of in vitro matured oocytes of Korean Native Cattle. Oocytes collected from slaughterhouse ovaries were matured in TCM 199 containing FSH, estradiol-17$\beta$ and FBS with granulosa cell monolayer for 24 hours and denuded with hyaluronidase. And then cumulus-free oocytes were submitted to a DC field of 1.0 kV/cm for 60 $\mu$sec in electroporation media(0.28 M mannito' and PBS) with different $Ca^2$+ concentations (0.00, 0.05, 0.10 and 0.15 mM). Stimulated oocytes were stained and examined for pronuclear formation after incuhation in SOF for 12 hours. The rates of pronuclear formation in hovine oocytes electrically stimulated in 0.28 M mannitol with 0.05, 0.10 and 0.15 mM $Ca^2$+(60.3, 82.2 and 75.0%) were significantly higher than without $Ca^2$+(6.3%) at 12 hours after an electric pulse(p<0.005). The activation rates of Korean Native Cattle oocytes stimulated in PBS supplemented with 0.05, 0.10 and 0.15 mM $Ca^2$+(71.0, 75.8 and 75.4%) were significantly higher than without $Ca^2$+(23.5%) after post-stimulation incubation(p<0.005). After incubation of oocytes in SOF with and without $Ca^2$+ following electric stimulation in 0.28 M mannitol with 0.10 mM $Ca^2$+, the rates of pronuclear formation of bovine oocytes in $Ca^2$+-free SOF(85.7%) was significantly higher than in SOF with 1.71 mM $Ca^2$+(62.5%, p<0.05). When oocytes were stimulated in two electrostimulation media supplemented with $Ca^2$+ and incubated in $Ca^2$+-free SOF, there were no significant differences in the rates of pronuclear formation hetween 0.28 M mannitol and PBS. These results indicate that a single electric pulse could induce activation of Korea Native Cattle oocytes in 0.28 M mannitol and PBS supplemented with $Ca^2$+. Furthermore, to improve the activation rates, it was hetter that stimulated oocytes were incubated in $Ca^2$+-free SOF after electric stimulation than in SOF with $Ca^2$+.

• Journal of Embryo Transfer
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• v.12 no.2
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• pp.181-188
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• 1997

This study was carried out to determine the effect of bovine follicular fluid(bFF), hormones, and fetal bovine serum(FBS) supplemented in the medium on the in vitro fertilization and development of bovine embryos. The ovaries were obtained from a local abattoir and placed in physiological saline kept at 30~32˚C and brought to the laboratory within 3~4 hours. The oocytes and follicular fluid were collected by aspiration from visible follicles, and the oocytes of grades I on the basis of the morphology of cumulus cells attached and the homogeneity of cytoplasmic granules were selected and used for maturation. The basal media used for oocyte maturation, fertilization and embryo development in vitro were Ham' F-10, TALP and TCM-199, respectively. The hormones supplemented in maturation medium were consisted of 35 pg /ml FSH, 10 pg /ml LH and 1 pg/mi estradiol-l7$\beta$. The bFF collected from 5~9 mm follicles was centrifuged, filtered and inactivated by heat-treatment at 56˚C for 30 min. FBS also was inactivated with the same method and kept at -20˚C until use. The embryos were co-cultured with the monolayer of bovine oviductal epithelial cells at 39˚C under 5% $CO_2$ in air for 9 days. The results obtained were summarized as follows: The fertilization rate of oocytes was found 87.4% from 10% FBS and hormones treatment for IVM, and 37.1% of these TVF embryos were developed to blastocyst stage in 10% FBS groups. Compared with this control system, the fertilization rate was decreased significantly(P<0.05) in the maturation without either FBS or hormones. These IVF embryos were developed to morula stage at the similar rate, but to blastocyst at significantly(P<0.05) lower rate in the embryo culture with or without FBS supplementation. The fertilization rate(82.9%) in hormones and 10% inactivated bFF was similar with 10% FBS and hormone groups(87.4%), but decreased significantly(P<0.05) in 20 or 30% bFF (61.0 or 66.0%), respectively. In vitro developmental competence to blastocyst stage in 10% FBS and 20% inactivated bFF(37.1% and 31.4%) was higher than in 10 or 30% inactivated bFF(20.0 or 19.2%) or 10, 20 and 30% fresh bFF(19.1, 21.0 and 17.5%) The results indicated that the in vitro fertillzation and development rate of the embryos should be improved in 10% FBS or 20% inactivated culture system and 20% inactivated bFF might be available economically for bovine oocyte maturation and embryo culture instead of fetal bovine serum.

• Asian-Australasian Journal of Animal Sciences
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• v.13 no.10
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• pp.1359-1366
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• 2000

An experiment was carried out to study plasma levels of hormones and metabolites of crossbred Holstein cattle during late pregnancy (28 days pre partum), early lactation (30 days post partum), mid-lactation (120 days post partum) and late lactation (210 days post partum). Two breed types of Holstein $Friesian{\times}Red$ Sindhi (50:50 = 50%HF) and Holstein $Friesian{\times}Red$ Sindhi (87.5:12.5 = 87.5%HF) were divided into four groups of four animals each. Two groups of each breed were fed with either rice straw treated with 5% urea or pangola hay (Digitaria decumbens) as the source of roughage throughout the experiments. There were a substantial increases in the mean levels of total triiodothyronine ($T_3$), insulin and glucagon at the onset of lactation, and maintained in a high levels during lactation advance for all groups of experiments. The mean levels of prolactin and thyroxine ($T_4$) were not significantly different among groups of animals, but the plasma cortisol concentration was slightly higher in both groups of 50%HF in comparison with those of 87.5%HF animals. The mean levels of plasma growth hormone (GH) of both groups of 87.5%HF animals feeding on either hay or urea treated rice straw markedly rose in the early period of lactation and markedly reduced in mid- and late lactation. These changes were accompanied with changes of milk yield. In contrast to 50%HF animals, plasma GH levels were considerably higher in the late pregnant period than in the early period of lactation and it remained constant as its value at the early lactation throughout the experimental period. The high levels of both plasma progesterone and estradiol concentration significantly declined after parturition and remained low through lactating period. The plasma glucose level in the 50%HF animals feeding on either hay or urea treated rice straw was higher than the 87.5%HF animals in all periods of experiments. Changes in plasma FFA levels of both types of crossbred animals were depended on the endocrine status during late pregnancy and lactation. The levels of plasma FFA of 50%HF animals were significantly higher (p<0.05) than those of 87.5%HF animals during late pregnancy. Both plasma ${\beta}$-hydroxybutyrate and lactate concentrations were not affected by feeding on either hay or urea treated rice straw during late pregnancy and lactation. These data demonstrate that there were no differences in the physiological performances in the same crossbred animals fed either hay or urea treated rice straw. The 87.5%HF animal has the genetic potential for a high milk yield and homeorhetic adaptation for mammary function differed from 50%HF animals during periods of lactation. Altering lactation persistency in 87.5%HF is regulated mainly by chronically acting growth hormones through the period of lactation.

• Asian-Australasian Journal of Animal Sciences
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• v.28 no.11
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• pp.1573-1582
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• 2015

This study was to investigate the effect of soyabean isoflavones (SIF) on onset of puberty, serum hormone concentration, and gene expression in hypothalamus, pituitary and ovary of female Bama miniature pigs. Fifty five, 35-days old pigs were randomly assigned into 5 treatment groups consisting of 11 pigs per treatment. Results showed that dietary supplementation of varying dosage (0, 250, 500, and 1,250 mg/kg) of SIF induced puberty delay of the pigs with the age of puberty of pigs fed basal diet supplemented with 1,250 mg/kg SIF was significantly higher (p<0.05) compared to control. Supplementation of SIF or estradiol valerate (EV) reduced (p<0.05) serum gonadotrophin releasing hormone and luteinizing hormone concentration, but increased follicle-stimulating hormone concentration in pigs at 4 months of age. The expression of KiSS-1 metastasis-suppressor (KISS1), steroidogenic acute regulatory protein (StAR) and 3-beta-hydroxysteroid dehydrogenase/delta-5-delta-4 isomerase ($3{\beta}-HSD$) was reduced (p<0.01) in SIF-supplemented groups. Expression of gonadotropin-releasing hormone receptor in the pituitary of miniature pigs was reduced (p<0.05) compared to the control when exposed to 250, 1,250 mg/kg SIF and EV. Pigs on 250 mg/kg SIF and EV also showed reduced (p<0.05) expression of cytochrome P450 19A1 compared to the control. Our results indicated that dietary supplementation of SIF induced puberty delay, which may be due to down-regulation of key genes that play vital roles in the synthesis of steroid hormones.

• Asian-Australasian Journal of Animal Sciences
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• v.23 no.8
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• pp.1080-1088
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• 2010

This experiment was conducted to investigate the effects of dietary energy levels of gestating gilts on physiological parameters and reproductive performance for primiparous sows. A total of 40 F1 gilts (Large White${\times}$Landrace) were allocated to 4 treatments using a completely randomized design (CRD). Four different experimental diets contained 3,165, 3,265 3,365 and 3,465 kcal of ME/kg and each diet was provided to gilts at 2.0 kg/d during gestation. Consequently, energy intake of each treatment of gestating gilts was 6,330, 6,530, 6,730 and 6,930 kcal ME/kg, respectively. During the whole gestation period, body weight, fat mass gain and backfat thickness of gilts were increased in proportion to dietary energy levels (p<0.01). However, estimated protein mass gain of gilts was not affected by dietary energy level (p>0.10). At farrowing, the total number of pigs born per litter did not show any significant difference among treatments. However, the number of pigs born alive per litter in treatment 6,730 kcal ME/d was significantly higher than that of other treatments (p<0.05). Moreover, litter weight at birth was improved as dietary energy level was increased (p<0.05). Feed intake of sows during lactation tended to decrease as dietary energy level of gestation was increased, but litter weight gain was not affected by dietary treatment during the gestation period. Fat content in colostrum was higher as dietary energy level was increased during gestation. The concentration of blood estradiol-$17{\beta}$ was increased and was higher at the first trimester of gestation in 6,730 kcal ME/d treatment compared to other treatments. These results suggested that increased dietary energy level during gestation resulted in higher body weight and backfat thickness of sows. In addition, reproductive performance of the sow, such as litter weight at farrowing and the number of pigs born alive, was improved when 6,730 kcal of ME/d treatment diet was provided. Consequently, the NRC (1998) recommendation of energy for gestating gilts (6,015 to 6,150 kcal of ME/d) should be reevaluated to maximize reproductive performance because recent high-producing sows require much more energy to produce a large litter size and heavier piglets from the first parity.

• Proceedings of the KSAR Conference
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• 2001.03a
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• pp.82-82
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• 2001

본 연구는 한우에 있어서 인공수정후 수태율증진을 위한 기초자료를 확보할 목적으로 성숙 미경산 한우의 발정동기화를 위해 CIDR(EAZI-BREED CIDR Plus, USA)를 7일간 질내에 삽입하였고, CIDR처리 제 6일째에 PGF2$\alpha$(lutlyase, USA)제제 5 $m\ell$을 근육주사하여 발정이 유기된 92%의 개체를 인공수정을 실시하였다. 수태율증진을 위해 무처리구를 두었고, hCG처리구(Chorulon, Intervet, Netherlands)는 2000IU를 근육 주사하였으며, CIDR처리구에는 Estradiol-17$\beta$의 캡슐을 제거한 CIDR를 수정후 7일부터 일주일간 질내에 삽입 처리하여 각 처리당 15두씩 총 45두의 한우를 실험에 공시하였다. 발정이 유기된 개체는 인공수정후 30일까지 2일 간격으로 채혈하여 혈중 Progesterone, IGF, IGF-II 및 cortisol 농도를 RIA법으로 분석하였으며 수정 후 60일 이후에는 직장검사법으로 임신감정을 실시하였다. 수정후 수태율은 대조구와 CIDR 처리구에서는 평균 62%였으나 hOG처리구는 73%로서 대조구에 비해 유의적으로 수태율이 높게 나타났다. 이때의 혈중 progesterone농도를 분석한 결과, 대조구에 CIDR 처리구의 수정후 7일째부터 증가하기 시작하여 제 12일째에서 평균 5.5ng/$m\ell$까지 증가하다가 그 이후 재발정 예정일일 21일째에 다소 감소되다가 다시 증가되어 일정한 수준으로 유지되었다 그러나 hCG처리구의 경우 수정후 5일부터 progesterone이 증가하다가 제 15일까지 약 10ng/$m\ell$까지 높은 progesterone이 유지되었다. 이와 같은 결과로 보아 hCG는 혈중 progesterone농도를 증가시킴으로써 난소내 임신황체의 progestorone분비 능을 촉진시키는 작용을 하는 것으로 사료되나, 한편 혈중 IGF I과 IGF-II농도는 대조구, hCG처리구 및 CIDR 처리구간의 차이가 없이 수정 후 제10일까지는 다소 높은 수준이었으나 그 이후 감소되는 현상으로 혈중 progesterone농도와는 부의 상관관계를 보였다. 따라서 IGF-I과 IGF-II는 혈중 progesterone의 농도를 인위적으로 조절하거나 황체의 progesterone분비기능을 직접 조절하는 역할은 하지 않는 것으로 사료되며, 특히 초기 임신의 수태율에 직접적으로 영향이 없는 것으로 사료된다. 또한 혈중 cortisol농도는 처리간의 차이는 확인할 수 없으나 임신과 비임신간의 혈중 cortisol농도는 유의적으로 차이가 있는 것으로 나타났다. 본 실험의 결과로 인공수정이나 수정란이식 및 체세포복제 수정란뿐만 아니라 형질전환 수정란의 이식후 수태율을 증진시켜 첨단기술의 조기정착을 위해 이식 후 7일째나 인공수정 후 7일째 hCG제제를 근육주사로 간단하게 처리함으로써 수태율을 크게 개선될 수 있으며 이것은 난소의 황체기능 즉, 황체로부터 충분한 progesterone을 분비할 수 있는 내분비적 환경을 제공하는 것으로 사료된다.

• Reproductive and Developmental Biology
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• v.32 no.2
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• pp.117-121
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• 2008

Our previous study showed that transgenic (TG) pigs harboring human EPO (hEPO) gene have been shown to have reproductive disorders, including low pregnancy rates, irregular estrus cycle and low little size. To investigate these reasons, we assessed estrus behavior (standing response) and plasma $17{\beta}$-estradiol ($E_2$) level, which partly reflect reproductive function, during the estrus cycles after synchronization and superovulation by hormone treatments. Then, we analysed blood composition and expression of hEPO gene in TG pigs. Pigs were injected with PG600. After 10 days, pigs were fed with Regumate porcine for 6 days. Blood samples were collected from jugular vein. Analysis of blood composition and $E_2$ level were measured by Hemavet 950 and $E_2$ ELISA kit, respectively. And, the expression of hEPO gene in reproductive organs was quantitated by real-time RT-PCR. The percentage of estrus behavior in TG was significantly decreased. Hematocrit (HCT), hemoglobin (Hb) concentration and red blood cell (RBC) number were significantly higher in TG than wild type (WT). On the other hand, high expression of hEPO gene in TG was observed in the mammary gland as well as in the uterus. Moreover, plasma $E_2$ level was significantly higher in TG than WT. These results suggest that nonspecific expression of hEPO gene in the other organs of TG may affect blood composition and plasma $E_2$ level, thereby causing reproductive disorders.

• Analytical Science and Technology
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• v.12 no.1
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• pp.34-39
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• 1999

Estrone such as estriol and estradiol can not be determined by votammetric methods, because these are electrochemically inactive in the potential windows for mercury drop electrode. Nitro-derivatives of estrone are electro active and nitration of estrone is accomplished by heating the solution involving estrone and sodium nitrite in a water-bath at $100^{\circ}C$ for 30 min. Such nitro-derivatives are determined directly by voltammetry. The electrochemical behavior for nitrated estrone was investigated by cyclic voltammetry. The trace estrone was determinated by differential pulse adsorptive cathodic stripping voltammetry. Nitrated estrone gives a well defined voltammetric wave at ca. - 0.61 V (vs. Ag/AgCl electrode). The electrochemical reaction was irreversible process in sodium borate buffer at pH 11 and nitrated estrone was strongly adsorbed on the surface of mercury electrode. The optimal experimental conditions for the determination of nitrated esterone were found to be 0.05 M sodium nitrate, 0.01 M sodium borate, pH 11.0, and an accumlation potential of 0.10 V (vs. Ag/AgCl). The detection limit was as low as $1{\times}10^{-9}M$ for estrone with 2 min accumulation time.

• Clinical and Experimental Reproductive Medicine
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• v.25 no.3
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• pp.251-260
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• 1998

It is well known that the clinical test for responsibility of accurate fertilization capacity in male partners is very important to diagnose and treat the infertility. However, it has been reported that the traditional semen analysis cannot accurately predict fertilization and pregnancy potential. The present study was performed to evaluate the acrosomal reaction to ionophore challenge (ARIC) test as a prognostic indicator for fertilization of sperm and oocyte in an in vitro fertilization and embryo transfer (IVF-ET) program. From March 1996 to Februry 1997, 30 couples undergoing IVF program were allocated to this study group. All female partners in the study group were 35 years old or less and their serum level of basal follicle stimulating hormone (FSH) and estradiol $(E_2)$ were normal. All the male partners have normal parameters of semen analysis. The ARIC tests were performed on the day of ovum pick up and in vitro insemination in all the male partners. The controlled ovarian hyperstimulation (COH) using luteal long protocol of gonadotropin releasing hormone (GnRH) agonist was used in all couples for IVF-ET. The acrosomal reaction with $10{\mu}l$ of 10% DMSO was induced spontaneously in $10.1{\pm}9.8%$, and acrosomal reaction with calcium ionophore A 23187 was induced in $27.4{\pm}18.1%$, and the ARIC value was $17.4{\pm}16.2%$. There were no significant correlation between the ARIC value and the fertilization rate ($r^2$=0.044, p=0.268). There were also no significant correlation between the ARIC value and the percentage of the grade I, II embryos ($r^2$=0.046, p=0.261). On the basis of above results, it was suggested that ARIC test might not be a useful prognostic indicator for fertilization in IVF-ET in male partners with normal parameters of conventional semen analysis. We guessed that IVF-ET could be performed to the patients primarily without universal appilcation of ARIC test to all male partenrs, and if fertilization failure occurs, the micro assisted fertilization (MAF) such as intracytoplsmic sperm injection (ICSI) might be used as an alternative mode of treatment with acceptable success rate.