• Asian-Australasian Journal of Animal Sciences
• /
• v.30 no.3
• /
• pp.439-445
• /
• 2017

Objective: Production of alpha-1,3-galactosyltransferase (${\alpha}GT$)-deficient pigs is essential to overcome xenograft rejection in pig-to-human xenotransplantation. However, the production of such pigs requires a great deal of cost, time, and labor. Heterozygous ${\alpha}GT$ knockout pigs should be bred at least for two generations to ultimately obtain homozygote progenies. The present study was conducted to produce ${\alpha}GT$-deficient miniature pigs in much reduced time using mitotic recombination in neonatal ear skin fibroblasts. Methods: Miniature pig fibroblasts were transfected with ${\alpha}GT$ gene-targeting vector. Resulting gene-targeted fibroblasts were used for nuclear transfer (NT) to produce heterozygous ${\alpha}GT$ gene-targeted piglets. Fibroblasts isolated from ear skin biopsies of these piglets were cultured for 6 to 8 passages to induce loss of heterozygosity (LOH) and treated with biotin-conjugated IB4 that binds to galactose-${\alpha}$-1,3-galactose, an epitope produced by ${\alpha}GT$. Using magnetic activated cell sorting, cells with monoallelic disruption of ${\alpha}GT$ were removed. Remaining cells with LOH carrying biallelic disruption of ${\alpha}GT$ were used for the second round NT to produce homozygous ${\alpha}GT$ gene-targeted piglets. Results: Monoallelic mutation of ${\alpha}GT$ gene was confirmed by polymerase chain reaction in fibroblasts. Using these cells as nuclear donors, three heterozygous ${\alpha}GT$ gene-targeted piglets were produced by NT. Fibroblasts were collected from ear skin biopsies of these piglets, and homozygosity was induced by LOH. The second round NT using these fibroblasts resulted in production of three homozygous ${\alpha}GT$ knockout piglets. Conclusion: The present study demonstrates that the time required for the production of ${\alpha}GT$-deficient miniature pigs could be reduced significantly by postnatal skin biopsies and subsequent selection of mitotic recombinants. Such procedure may be beneficial for the production of homozygote knockout animals, especially in species, such as pigs, that require a substantial length of time for breeding.

• Korean Journal of Animal Reproduction
• /
• v.26 no.1
• /
• pp.79-85
• /
• 2002

We investigated the optimal concentration and exposure time of cycloheximide(CHX) on development of activated porcine oocytes following electrical pulse(EP). After 42~44 h maturation, oocytes were treated with 0.1% hyaluronidase, and denuded cumulus cells by pipetting. Oocytes were stimulated by electric pulse (1.2 kV/cm, 30 $\mu$sec, 1 pulse) or incubated for 3, 5 and 7 h in cycloheximide (1, 5 and 10 $\mu\textrm{g}$/$m\ell$, respectively) following electric pulse, and cultured for 8 days. Cleavage rate of oocytes activated with 10 $\mu\textrm{g}$/$m\ell$ CHX following EP was significantly (P＜0.05) higher than those of 1 $\mu\textrm{g}$/$m\ell$ (86.8% vs. 74.4%). The developmental competence of oocytes incubated to 5 $\mu\textrm{g}$/$m\ell$ of CHX was significantly (P＜0.05) higher development to blastocysts (13.3%), compared with 10 $\mu\textrm{g}$/$m\ell$ of CHX (5.6%). When the oocytes were activated with 5$\mu\textrm{g}$/$m\ell$ CHX for 3, 5, and 7 h following EP, the cleavage rate of oocytes in 5 h group(86.6%) was significantly (P＜0.05) higher than that in 3 h group(73.2%). The developmental rate of oocytes to morula in 5 and 7 h groups(26.7% and 16.4%) were significantly (P＜0.05) high than that in 3 h group(14.5%). Matured oocytes were activated with electric pulse (EP) or electric pulse combined with cycloheximide (EP + CHX) and cultured for 8 days. The rate of cleavage and development to blastocyst (80.1% and 11.6%) of activated with EP group were similar to EP combined with CHX group. When activated with EP or EP combined with CHX, the mean cell number of blastocysts were less in the activated with EP (18.67$\pm$5.53) than in the activated EP combined CHX (20.71$\pm$6.16), but not significantly different. This results suggest that, when the porcine oocytes were activated with CHX following EP, the developmental rate of activated oocytes can be improved by treated with a concentration of 5 $\mu\textrm{g}$/$m\ell$ CHX for 5 h exposure time.

• Korean Journal of Animal Reproduction
• /
• v.24 no.4
• /
• pp.357-364
• /
• 2000

Members of the glycoprotein family, which includes CG, LH, FSH and TSH, comprise two noncovalently linked $\alpha$- and $\beta$-subunits. Equine chorionic gonadotropin (eCG), known as PMSG, has a number of interesting and unique characteristics since it appears to be a single molecule that possesses both LH- and FSH-like activities in other species than the horse. This dual activity of eCG in heterologous species is of fundamental interest to the study of the structure-function relationships of gonadotropins and their receptors. CG and LH $\beta$ genes are different in primates. In horse, however, a single gene encodes both eCG and eLH $\beta$ -subunits. The subunit mRNA levels seem to be independently regulated and their imbalance may account for differences in the quantities of $\alpha$ - and $\beta$-subunits in the placenta and pituitary. The dual activities of eCG could be separated by removal of the N-linked oligosaccharide on the $\alpha$-subunit Asn 56 or CTP-associated O-linked oligosaccharides. The tethered-eCG was efficiently secreted and showed similar LH-like activity to the dimeric eCG. Interestingly, the FSH-like activity of the tethered-eCG was increased markedly in comparison with the native and wild type eCG. These results also suggest that this molecular can implay particular models of FSH-like activity not LH-like activity in the eCG/indicate that the constructs of tethered molecule will be useful in the study of mutants that affect subunit association and/or secretion. A single-chain analog can also be constructed to include additional hormone-specific bioactive generating potentially efficacious compounds that have only FSH-like activity. The LH/CG receptor (LH/CGR), a membrane glycoprotein that is present on testicular Leydig cells and ovarian theca, granulosa, luteal, and interstitial cells, plays a pivotal role in the regulation of gonadal development and function in males as well as in nonpregnant and pregnant females. The LH/CGR is a member of the family of G protein-coupled receptors and its structure is predicted to of a large extracellular domain connected to a bundle of seven membrane-spanning a-helices. The LH/CGR phosphorylation can be induced with a phorbol ester, but not with a calcium ionophore. The truncated form of LHR also was down-regulated normally in response to hCG stimulation. In contrast, the cell lines expressing LHR-t631 or LHR-628, the two phosphorylation-negative receptor mutant, showed a delay in the early phase of hCG-induced desensitization, a complete loss of PMA-induced desensitization, and an increase in the rate of hCG-induced receptor down-regulation. These results clearly show that residues 632~653 in the C-terminal tail of the LHR are involved in PMA-induced desensitization, hCG-induced desensitization, and hCG-induced down-regulation. Recently, constitutively activating mutations of the receptor have been identified that are associated with familial male-precocious puberty. Cells expressing LHR-D556Y bind hCG with normal affinity, exhibit a 25-fold increase in basal cAMP and respond to hCG with a normal increase in cAMP accumulation. This mutation enhances the internalization of the free and agoinst-occupied receptors ~2- and ~17- fold, respectively. We conclude that the state of activation of the LHR can modulate its basal and/or agonist-stimulated internalization. Since the internalization of hCG is involved in the termination of hCG actions, we suggest that the lack of responsiveness detected in cells expressing LHR-L435R is due to the fast rate of internalization of the bound hCG. This statement is supported by the finding that hCG responsiveness is restored when the cells are lysed and signal transduction is measured in a subcellular fraction (membranes) that cannot internalize the bound hormone.

• Korean Journal of Animal Reproduction
• /
• v.23 no.1
• /
• pp.1-12
• /
• 1999

The present study was conducted to find the effects of different cadmium(Cd) levels in diets on clinical toxicity, sperm capacity and histopathological changes in rats. Thirty male rats of Sprague-Dawley weighing 125.3$\pm$15.2g were randomly blocked into five groups according to body weights. Five levels of Cd in AIN-76 purified diet(0, 25, 50, 100 and 250 ppm) had been fed for 8 weeks. Cadmium was supplemented with a form of CdCl$_2$. 1. After 8 weeks of Cd intake had resulted in apparent cadmium intoxication; reduced growth rate, enlarged kidney and testis, decreased hematocrit value and hemoglobin content in response to supplemented Cd levels in the diets. 2. Cadmium accumulation in liver and kidney showed a tendency to increase in cadmium-exposed groups. The levels of metallothionein were also significantly elevated in the tissues of liver in response to the levels of Cd supplemented(P＜0.05). 3. Although sperm motility was not significantly different among treatments, rats fed Cd tended to have reduced sperm motility but sperm concentration of Cd supplemented groups were significantly lower than that of control(p＜0.05). 4. Based on the findings from gross lesion, rats fed 250ppm of Cd were externally emaciated, had exposed penis and observed atrophies of kidney and testis. Histopathological observation seemed that the liver of groups feeding Cd supplemented diets showed cellular degeneration and accumulation of eosinophilic materials in the capillaries. In kidney, rats fed Cd diets had shown tubular epithelium degeneration and lesions of basophilic materials, while testes were weakened in numbers of spermatid and sporadically enlarged of giant cells.

• Korean Journal of Animal Reproduction
• /
• v.23 no.1
• /
• pp.75-84
• /
• 1999

This study assessed development in vitro of pronuclear(PN) stage embryos cryopreserved by the method of either vitrification or slow freezing, by using of different cryoprotectants, and equilibration and cooling rate, in rabbit. Ethyleneglycol- ficoll- sucrose(EFS) or ethyleneglycol- polyvinylpyrrolidone - galactose- (EPG-I) for vitrification, and EPG- II for slow freezing as cryoprotectant were used. The pronuclear embryos were exposed to EFS for 0 to 5 min and diluted with D-PBS and/or pre-dilution with 0.5 M sucrose. To examine the viability of frozen-thawed embryos, PN embryos were co-cultured with bovine oviductal epitherial cell(BOEC) for 5 days to hatching blastocyst stage in 39 $^{\circ}C$ 5% $CO_2$incubator. The results obtained were as follows: The dilution with 0.5 M sucrose and D-PBS after the exposure to EFS for 1.0 min resulted in no significant(P＜0.05) decrease in the development of PN embryos to hatching blastocyst(72.0%), compared with controls. The development of PN embryos cryopreserved to hatching blastocyst was not significantly (P＜0.05) different between EFS for 1.0 min(72.0%), EPG-I for 1.0 min(72.0%) and EPG-II for 30 min(66. 7%). The post-thaw development of PN embryos to hatching blastocyst was similarly very low as 6.1% and 11.5% in vitrification with EFS and slow freezing with EPG-II, respectively. The incidence of post-thaw zona-crack in PN embryos cryopreserved by slow freezing with plunging to liquid nitrogen at －35$^{\circ}C$ was signicantly(P＜0.05) higher(25.0%), compared with －85$^{\circ}C$ (1.9%). These results indicated that the rabbit PN embryos could be cryopreserved with either vitrification or slow freezing procedure, and frozen PN embryos could be successfully developed in vitro to haching blastocyst. but the post-thaw development of cryopreserved PN embryos was still very low under the present conditions.

• Korean Journal of Animal Reproduction
• /
• v.22 no.1
• /
• pp.1-9
• /
• 1998

This study was carried out to confirm whether the developmental capacity of bovine mature oocytes frozen ultra-rapidly using electron microscopic(EM) grids and EFS30 can be obtained, and whether the cryoprotectants and the freezing method used in this study effect detrimentally to the bovine oocytes by indirect immunocytochemistry. As freezing solution, we used EFS30 which consisted of 30% ethylene glycol, 0.5 M sucrose, 18% ficoll and 10% FBS added in D-PBS. The results obtained in this experiment were summarized as follows: When the effects of cryoprotectant and freezing procedure on the microtuble, micrfilament and chromatin morphology of oocytes were evaluated using indirect immunocytochemistry, the results of freezing as well as exposure group were not different with that of the control oocytes. When the fertilization abnormality after ultrarapid freezing of bovine mature oocytes was examined by Hoechst staining, the rates of total penetration(96.7, 9.0%), normal two pronuclei formation(74.6, 68.9%) and mean number of sperm / oocyte(1.50, 1.44) were not different between control and freezing group. In addition, when the developmental capacity of frozen-thawed of oocytes(85.5%) was survived, 74.5% of them were cleaved and 31.4% of cleaved embryos were developed to blastocyst. These data were similar to those of the control(76.0%, 34.6%) and exposure(74.5%, 33.0%) except survival rates. Also, when the total cell number of blastocysts produced from the each treatment at day after IVF was examined by hoechst staining, there were not different among groups. There results demonstrate that developmental capacity of frozen-thawed bovine mature oocytes can be successfully obtained by ultra-rapid freezing method using EM grid and EFS30 solution.

• Journal of Animal Environmental Science
• /
• v.1 no.1
• /
• pp.9-19
• /
• 1995

This study was carried out to investigate the effect of sawdust bedding barn on manure handing, management labour and physiological aspect of dairy cows, and then to establish the criteria on the optimum utilization method of sawdust bedding barn. 46 tie stall barns and 49 sawdust bedding barns were surveyed to cmopare the milk productivity between two different barns, and also 5 tie stall barns, 15 sawdust bedding barns and 1 rice hull bedding barn were selected to study the utilization situation of sawdust barn in Kyung-Ki province area. The major results obtained were as follows; 1. The roof material of sawdust barn were consisted of 66% vinyl house, 23% PVC light and 11% slate and galvanum coated tin. Most of the floor structure was earth ground with the rate of the approximately 82%. 2. The average occupied area of sawdust bed per cow was 15.2 $m^2$, depth that 30 cm and the utilization period was 12 months. 3. Milk Yield was significantly higher at sawdust barn than at tie stall barn(P<0.01). Bacterial and somatic cell count in raw milk were less at the sawdust barn than in tie stall barn. However, there was not significance difference between two barns. 4. The labour hour needed to cow management in the sawdust barn was approximately 48% of that of tie stall barn. 5. The temperature and moisture content measured in sawdust bed were closely affected by seasonal ambient temperature. The skin and hair of cow were much cleaner at the PVC light roofed sawdust barn than any other roof materials. 6. The additives used for improving of fermentation did not show any effect on temperature and moisture content in sawdust bed. When the ambient temperature was $30.4^{\circ}C$, the surface temperature of measured 1 cm above the sawdust bed was $12.2^{\circ}C$ lower and the temperature of 100 cm above the sawdust bed was $2.4^{\circ}C lower under shading net facility than that of vinyl roofed one.7. The hoof length of miking cow was 7.95 cm in tie stall and 9.19 cm in sawdust barn with high significance (P<0.01). And disease occurence such as mastitis and foot-rot tended to decrease in the sawdust barn. 8. The number of total bacteria and coliform bacteria were less in the sawdust bed compared with earth ground resting area. And a parasite strongyloides papillosus was detected but without any infected cow. 9. The nitrate($NO_3N$) content in non-roofed earth ground resting area and earth ground under the sawdust bed was likely to pollute the ground water. 10. In economic point of view, rice hull bedding barn was the cheapest among different systems. And in the sawdust bedding barn PVC light + slate roofed barn was most desirable, and vinyl roofed one the least.

• Korean Journal of Animal Reproduction
• /
• v.22 no.3
• /
• pp.293-300
• /
• 1998

The objective of this study was to determine effects of $\beta$-mercaptoethanol ($\beta$-ME) and cyst-eine (CYS) on the development of bovine em-bryos obtained from in vitro matured and fertil-ized oocytes. Cumulus-oocyte-complexes (COC-s) were matured in micro-drop of TCM-199 medium containing 10% FBS, 17$\beta$-Estradiol and FSH-p under paraffin oil at 39$^{\circ}C$ for 24 hrs. The fertilization of COC were induced in Fert-TALP medium supplemented with PHE, heparin, BSA and then the fertilized oocytes were cultured in CR1aa medium for 24 hrs. To investigate the effects of the agents on the development of the embryos, the embryos developed to the late 2-cell stage were cultured in the media with and without $\beta$-ME, CYS for 9 days. In experiment 1, to select appropriate concentration of $\beta$-ME and CYS during whole culture period (9 days), various concentrations of $\beta$-ME and CYS were add ded to the CR1aa medium. Addition of 25TEX>$\mu$M of $\beta$-ME and O.1mM of CYS to the culture medium 1 increase the incidence of embryos developed to the blastocyst. In experiment 2, we evaluated the effects of 25$\mu$M of $\beta$-ME and O.1mM of CYS addition on the blastocyst formation when emb bryos at different stages were exposed to 25$\mu$M $\beta$-ME and O.1mM of CYS. $\beta$-ME and CYS enhanced in vitro development of embryos to the blastocyst stage. The effect was greater in 8-ceII to morula embryos than in embryos fewer than 2-cells at the initiation of treatment. These results suggested that the addition of 25$\mu$M B-ME and O.1mM cysteine enhanced development to the blastocyst and hatching stage of in vitro derived bovine embryos, also addition of $\beta$-ME and cysteine were effective later stage embryo than early embryo development.

• Korean Journal of Animal Reproduction
• /
• v.25 no.2
• /
• pp.163-169
• /
• 2001

OSBA(oocytes-sperm binding assay) is a tool developed for rapid test of optimal condition of IVF medium and protein source by binding ability of mouse sperm and egg. Mouse oocyte-cumulus complexes were prepared by removing of the cumulus cells with 0.1% hyaluronidase. 10$\pm$2 oocytes per 30 ${mu}ell$ medium drop were inseminated with 3 ${mu}ell$ sperm suspension and were cultured f3r 3 hours and 24 hours, respectively. And the oocytes were recovered gently and the No. of sperm bound on oocytes were counted. In the Exp. 1, the ratio of oocytes bound with one sperm at least were 60.2%(50/83), 2%(2/77) and 100%(79/79) in the medium with no protein, FBS(15%, v/v) and BSA(0.4%. w/v), respectively, Fetal bovine serum(FBS) seriously inhibited sperm binding on oocyte, although bovine serum albumin(BSA) promoted the binding ability. The inhibiting effect of FBS was dependent on the concentration of FBS. The sperm binding ability according to oocyte maturity was tested in the Exp. 2. There was no significant difference between Met. II (mature) and Met. I (intermediate mature) oocytes in the number of oocytes bound with sperm and the number of sperm bound on oocytes. Finally, in Exp. 3, two batches of Ham's F10 medium with good and poor quality by OSBA were tested (The ratios of embryos developed from PN 1-cell stage to hatched blastocyst; 25% vs. 70%). In the medium with good quality, sperm binding ability was significantly increased (P ＜ 0.05). The ratio of oocytes bound with one sperm at least was 66% and 90% in the medium with poor and good quality, respectively. Conclusively, It was possible to test IVF medium condition rapidly and easily by OSBA.

• Journal of Animal Science and Technology
• /
• v.48 no.2
• /
• pp.293-306
• /
• 2006

We characterized physicochemical properties and examined the organoleptic and textural evaluations of Feta cheese made from goat's milk. Nutritional compositions of goat Feta cheese were fat 23.50%, protein 11.03% with moisture content of 59.54%. Cell numbers of lactic starter cultures in Feta cheese maintained from log 8.46 CFU/g and pH 5.76 during storage at 4℃ for 14 day's aging. The color of Feta cheese was whitish (L. 93.19) at after finishing brine salting, but became a little yellowish(b. 3.52) (a. -0.71). For texture profile analysis of goat Feta cheese, hardness, fracturability springness, and cohesiveness seemed to be week, but adhesiveness gumminess, chewiness, and resilience were enhanced as aging times extended to 14days, resulted in the overall textural properties was to be superior to control cheese(commercial Mozzarella cheese). Organoleptic evaluations were examined based on the intensities and the preferences for flavour, tastes, texture and mouth feeling. saltiness, bitterness and acidity were stronger in the intensities than control cheese, but the preferences were enhanced by aging to be better than control cheese at 14 days and later on, however, the texture changed to be weaker in hardness and unpleasant in mouthfeel. The fatty acid compositions of Feta cheese analysed by Gas chromatography were saturated fatty acid 42.06%, monoenoic acids 29.67%, di-enoic acids 24.24%, tri-enoic acids 1.21%.