• Journal of Aquaculture
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• v.21 no.4
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• pp.285-293
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• 2008

Vitellogenin (Vg) is the precursor of vitellin (Vn), the major yolk protein of teleost fishes. In this study, Vg and Vn proteins of the Korean bullhead Pseudobagrus fulvidraco were isolated using gel-filtration chromatography (Sephadex-G 200 column) and anion-exchange chromatography (Mono Q HR 5/5 column), respectively. Purified Vn with an estimated molecular mass of 360 kDa by gel filtration chromatography was obtained from ovarian egg, and it was composited to one major subunit with an estimated molecular mass of 107 kDa by SDS-PAGE. In the result of western blotting, one major band was detected using antiserum against Vn (anti-Vn). These results suggested that Vn was composed of three subunits having the same molecular weight in Pseudobagrus fulvidraco. Vg was induced by estradiol-$17{\beta}$ ($E_2$) and purified from $E_2$ treated male serum. The molecular weight of whole Vg was estimated to be 450 kDa by gel filtration chromatography, and it is composed of three subunits with estimated molecular masses of 110 kDa, 125 kDa and 147 kDa as determined by SDS-PAGE. In the Ouchterlony's immunodiffusion test using anti-Vn and antiserum against female and male serum, purified Vg was detected in matured female and Ez treated male serum but not in untreated male. These results can be used in detecting estrogenic contamination of the aquatic environment.

• Korean Journal of Fisheries and Aquatic Sciences
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• v.34 no.1
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• pp.13-16
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• 2001

Plasma levels of sex steroid hormones in rockfish, Sebastes inermis were examined monthly in relation to gonadosomatic index (GSI) under a controlled water temperature and photoperiod, The GSI of a control group (C) in female began to increase from November and reached a maximum in January, Sample fish under a controlled water temperature and photoperiod (Tr) were divided into a responded group (Tr-r) and a un-responded group (Tr-n) by the gonadal maturation condition and GSI. The GSI of females in Tr-r reached a maximum in March. But the female GSI in Tr-n kept lower than 1.2 during the experimental period. No differences in male GSI were noticed between C and Tr. The $estradiol-17\beta$ and testosterone levels of female plasma in Tr reached a maximum in October, later than those in C. In males, these was no difference in 11-ketotestosterone and testosterone between C and Tr. When rockfish was reared in September under the controlled water temperature and photoperiod which were equivalent to those in July, that is two months earlier, the maturation of females was delayed in comparison with C. This finding suggested that delayed maturation in ovary was caused by the secretion of sex steroid hormones in relation to the water temperature and photoperiod of the hypothalamus-pituitary-gonad axis.

• Journal of Embryo Transfer
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• v.24 no.3
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• pp.199-205
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• 2009

This study was carried out to investigate the effective genetic resources preservation system using the frozen boar semen. The porcine oocytes were matured for 44 hours in NCSU-23 medium with or without 10% Porcine Follicle Fluid (PFF), 0.5 ${\mu}g/ml$ porcine FSH, 0.5 ${\mu}g/ml$ equine LH, 1.0 ${\mu}g/ml$ 17 $\beta$-estradiol ($E_2$) and 10 ng/ml Epidermal Growth Factor (EGF) under mineral oil at $38.5^{\circ}C$ in humidified atmosphere of 5% $CO_2$ in air. After 44 h of culture, the oocytes were inseminated with frozen-thawed semen and fresh semen prepared with mTBM medium for 6 h. Later, set of 50 presumptive zygotes were transferred into 4-well dish (500 ${\mu}l$) of IVC medium. for embryos freezing, slow-freezing and vitrification methods were used as a cryopreservation. Differences among treatments were analyzed using General Linear Model Procedure by SAS Package (version 6.12) differences were considered significant when p<0.05. Following IVF and IVC, the rates of cleavage and blastocysts formation were significantly higher (p<0.05) in hormone supplemented group than that of hormone-free group (25.7 vs, 12.1). The development rates to cleavage and blastocysts were significantly higher in PZM-5 group than NCSU-23 group (60.3%, 46.6% vs 27.4%, 11.1%). Further improvement was achieved when PZM-5 was supplemented with FBS. Cleavage rates was significantly higher in fresh semen source group than frozen semen (66.7% vs 43.7%). However in blastocysts rates was similar two groups. Post-thaw survival rates of embryos were 1.2% and 2.2% in slow-frezing and vitrification groups, respectively. The results of our study suggest that it is still possible to improve the culture conditions and boar semen cryopreservation for enhance reproductive technology and animal genetic resources conservation.

• Journal of Embryo Transfer
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• v.29 no.1
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• pp.35-41
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• 2014

Laparoscopic ovum pick-up (LOPU) is a convenient method for collecting oocytes in small ruminants. LOPU has the advantage of being a less invasive means of oocyte collection, thereby allowing for a repeated usage of the oocyte donor animals. A total of 25 Korean black goats were used in the winter season (December to February) and LOPU was applied to the goats which had been treated for superovulation more than two times during the last twelve months. Estrus was synchronized with an intravaginal insert containing 0.3 g progesterone for 10 to 12 days. Ovaries were hyperstimulated with eCG 1,000 IU oneshot, FSH with eCG (50 mg / 1,000 IU; 70 mg / 500 IU; 70 mg / 1,000 IU) oneshot or FSH multiple-shot with eCG oneshot ($20mg{\times}6/300IU$) given intramuscularly 72 h prior to LOPU. For these groups, the number of follicles (mean ${\pm}$ SEM) observed which developed to larger than 2 mm in diameter were $1.6{\pm}2.5$, $4.3{\pm}3.1$, $5.5{\pm}4.2$, $6.6{\pm}2.1$ and $8.8{\pm}7.8$, respectively. Oocytes were aspirated by using OPU needles and a vacuum pump. The overall oocyte retrieval rates were 41.4%. Oocytes were matured in TCM-199 supplemented with 10% (w/v) bovine serum albumin + $10{\mu}g/ml$ FSH + $1{\mu}g/ml$ $17{\beta}$-estradiol for 27 h at $39^{\circ}C$ in 5% $CO_2$ in air. Oocytes were parthenogenetically activated by ionomycin combined with 6-diethylaminopurine (6-DMAP). Total oocyte maturation and cleavage rate were 67.3% and 78.8%, respectively. In summary, LOPU is a useful oocyte collection method in Korean black goats that can provide immature oocytes for transgenesis or nuclear transfer.

• Journal of Animal Science and Technology
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• v.62 no.4
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• pp.521-532
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• 2020

The production performance of broiler breeder hens in response to different levels of total lysine during the early laying period was investigated. A total of 126 Ross 308 parent stock hens were offered one of seven dietary treatments formulating elevated contents of total lysine ranging from 0.55% to 0.79% (0.04 scale; 133 g of feed) from 23 to 29 weeks of age. Each treatment had six replicates with three birds per pen. Body weight was recorded triweekly and eggs were collected and weighted at 9:00 am daily. One hen from each pen was euthanized to collect blood samples and visceral organs were harvested and weighed. Egg production, egg weight and egg mass were lower (p < 0.05) in hens offered a diet containing 0.55% total lysine compared to those fed the diet containing higher total lysine. Hens offered a diet containing 0.71%, 0.75%, and 0.79% total lysine had greater (p = 0.008) egg production rate compared to those offered a diet containing lysine less than 0.71%. The number of total eggs produced tended to be greater (p = 0.083) in hens offered a diet containing 0.71 and 0.75% total lysine compared to the other treatments. The number of settable egg production was higher (p < 0.001) in hens offered a diet contacting 0.79% total lysine compared to those fed the diet containing lower levels of total lysine. The relative weights of oviduct and ovary were lower (p < 0.05) in hens offered a diet containing 0.59% total lysine compared to the other treatments. No difference found in body weight, the number of total eggs, double-yolk eggs and abnormal shell eggs among the treatments. The urea nitrogen, estradiol-17 beta and progesterone in plasma were not affected by treatments. Based on linear- and quadratic-plateau models, total lysine requirements for egg production, settable egg production and egg mass at the early laying period were to be 0.73%, 0.77%, and 0.71%, respectively. Modern broiler breeder hens likely require higher total lysine than NRC recommendation in a diet for enhancing productivity during the early-laying period.

• Toxicological Research
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• v.27 no.3
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• pp.181-184
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• 2011

Screening of estrogenic activity on dichloro diphenyl trichloroethane (DDT), dichloro diphenyl dichloro ethylene (DDE), dieldrin, heptachlor, aldrin, chlordane, lindane, polybrominated diphenyl ethers (PBDE) and parabens was compared using Organization for Economic Cooperation and Development (OECD) test guideline 455 (TG455). The estrogenic activity of DDT was 58,000-fold ($PC_{50}$, $1.67{\times}10^{-6}$ M) less than $17{\beta}$-estradiol($E_2$) ($PC_{50}$, $2.88{\times}10^{-11}$ M) but DDE, dieldrin, heptachlor, aldrin, chlordane, lindane and PBDE did not show any estrogenic activity in this assay system. In the case of paraben compounds, the rank of relative transcriptional activation (logRTA) was butyl paraben -1.63752 ($PC_{50}$, $1.25{\times}10^{-7}$ M) > isobutyl paraben -2.34008 ($PC_{50}$, $6.3{\times}10^{-7}$ M) > ethyl paraben -2.64016 ($PC_{50}$, $1.26{\times}10^{-6}$ M) > isopropyl paraben -2.73993 ($PC_{50}$, $1.58{\times}10^{-6}$ M) > propyl paraben -2.84164 ($PC_{50}$, $2.0{\times}10^{-6}$ M). Our data suggest that OECD test guideline TG455 may be useful as a screening tool for potential endocrine disruptors.

• 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.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.