• Reproductive and Developmental Biology
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• v.36 no.3
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• pp.147-154
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• 2012

This study was designed to determine whether low-density lipoproteins (LDL) from egg yolk and taurine, hypotaurine and trehalose as antioxidant in extender improve the freezability and fertility of Korean Jeju Black Bull semen. The semen was cryopreserved with tris egg yolk extenders containing 7% glycerol and treated 4% LDL, 20 mM taurine, hypotaurine and trehalose. Frozen-thawed sperm were evaluated motility, viability, membrane, and acrosome integrity and sperm penetration ability. The results were compared to semen cryopreserved in tris egg yolk extender only as control. Frozen-thawed semen evaluation cleary indicated that the addition of LDL and LDL-antioxidants (taurine, hypotaurine and trehalose) combination were significantly improved (p<0.05) the viability (%; with staining test using eosin-Y) compared to control spermatozoa. Also, in membrane integrity (%; with supravital hypo-osmotic swelling test), not only LDL-antioxiants combination but also LDL were significantly increased (p<0.05) the swelled sperm using HOST compared to control. Sperm acrosome integrity state was classified by CTC (chlortetracycline) staining test. F pattern was significantly increased in LDL-antioxidant combination than control (p<0.05) and B pattern was not significantly differences among all treatments and control. However, AR pattern was significantly decreased in LDL-antioxidants combination than control (p<0.05). Pronucleus formation and sperm penetration index (SFI) were significantly increased in LDL and LDL-antioxidants combination than control (p<0.05). Especially, LDL-taurine significantly improved pronucleus fomation and SFI than LDL (p<0.05). It was concluded that LDL and LDL-antioxidants in extender improved the freezability and fertility of Korean Jeju Black bull spermatozoa.

• Korean Journal of Animal Reproduction
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• v.20 no.2
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• pp.127-134
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• 1996

The objective of this experiment was to test the ability of the fertilization of EGF treated pig oocytes for in vitro maturation. The addition of EGF (10 ng/ml), FSH (10 ${\mu}\textrm{g}$/ml), or FBS (10%) on maturation medium of pig immature oocytes divided into four groups as follows; group 1: untreatment, group 2: EGF alone, group 3: combination of FSH and FBS, or group 4: combination of EGF, FSH, and FBS. The interactive effects of nuclear maturation rates (M II%) of EGF alone, FSH plus FBS, and EGF plus FSH added FBS treatments were significantly higher than those of non-treatments (P<0.001). The fertilization rate of EGF alone (group 2) was lower than that of 3, 4 groups, but was significantly higher than group 1 (p< 0.005). Furthermore, combination of EGF, FSH,and FBS (group 4) was higher than others (group 1. 2, 3) on male pronuclei formation as well as penetration of sperm (P<0.05). These results suggested that EGF alone decreased the ability of cytoplasmic maturation compared to nuclear maturation in pig oocytes, but a high level of cytoplasmic maturation of in vitro-matured pig oocytes can be achieved when supplemented with FSH and FBS.

• Journal of Embryo Transfer
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• v.16 no.2
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• pp.91-98
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• 2001

We evaluated the effects of the substrate and inhibitors related to phosphatidylinositol metabolism on in vitro maturation and fertilization of porcine oocytes. Cumulus-oocyte complexes were cultured in mTLP-PVA medium supplemented with or without inositol (250 mM) fur 46h. Subsequently, these oocytes were inseminated with fresh boar semen in mTALP-PVA medium for 6h. At 6h after insemination, oocytes were cultured for further 12 h in TCM-199 supplemented with 10% FBS (fetal bovine serum). The higher percentage of oocytes in inositol-supplemented medium reached metaphase of the second meiotic division compared to those in control (81.4% vs. 67.3%; P<0.()5). following 18 h of insemination, more number of male pronuclei were formed in the oocytes matured in inositol-supplemented medium than in those of control experiment (42.0% vs. 27.3%; P<0.05). When oocytes were cultured in medium with 10mM LiCl (chloride lithium) or 0.5mM dbcAMP (dibutyryl cyclic adenosine monophosphate) to determine the role of inositol on the maturation of oocytes, these two drugs inhibited the meiotic division of oocytes (P<0.05). However, addition of inositol to the culture medium did overcome the inhibitory effect of these drugs on the oocyte maturation. DbcAMP and verapamil supplemented synergistically arrested the meiotic division of oocytes. Addition of verapamil did not inhibit germinal vesicle breakdown, but it severly inhibited the second meiotic division of oocytes. These results suggest that inositol exert its improving effects on maturation, by activating the PI (phosphatidylinositol) cycle and causing beneficial changes in both cytoplasm and membrane of oocytes.

• Korean Journal of Animal Reproduction
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• v.23 no.1
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• pp.19-27
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• 1999

This study was carried out to investigate the effect of intracytoplasmic injection of $Ca^{2＋}$ and human sperm extract on the parthenogenetic activation of mouse oocytes. The results obtained were as follows: 1. The mouse oocytes were injected with 10 pl of PBS medium containing 0, 1.7 and 5 mM calcium concentrations, respectively. Activation rate of the oocytes with formation of pronuc1ei and extrusion of the second polar bodies was 14.5, 9.8 and 14.9% at the above calcium concentrations, respectively. There were no significant differences in the activation rates among the calcium concentrations. 2. The mouse oocytes were injected with 10 pl of non-heated human sperm extract, and cultured for 12~15 h in the PBS media with the 0, 1.7 and 5 mM calcium concentrations, respectively. Activation rate(51.8%) of the oocytes at the 1.7 mM calcium concentration was significantly higher than those at the 0 and 5 mM calcium concentrations. 3. The mouse oocytes were injected with 10 pl of heated human sperm extract, and cultured for 12~15 h in the PBS media with the 0, 1. 7 and 5 mM calcium concentrations, respectively. No significant differences were found in the activation rates (11.8~17.0%) among the calcium concentrations. 4. The mouse oocytes were injected with 10 pl of PBS medium, non-heated sperm extract and heated sperm extract, and cultured for 12~15 h in the PBS media with 1.7 mM calcium concentrations, respectively. Activation rate (54.5%) of the oocytes injected with the non-heated sperm extract was highest. There were significant differences in the activation rates among the above injection materials (P＜0.05). 5. The mouse oocytes were injected with 10 pl of 1 and 6 days old non-heated sperm extracts, and cultured for 12＜15 h in the PBS media with 1.7 mM calcium concentrations, respectively. Activation rate(60.0%) of the oocytes injected with 1 days old sperm extract was significantly higher than that (11.1%) injected with 6 days old sperm extract. The results obtained in this study suggest that non-heated human sperm extract may contain sperm-associated oocyte-activating factor such as oscillin.

• Journal of Embryo Transfer
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• v.19 no.2
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• pp.173-183
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• 2004

This study was carried out to examine the effects of epidermal growth factor (EGF) and the number of cumulus-oocyte complexes (COCs) on in vitro maturation (IVM) of porcine immature oocytes, and on subsequent in vitro fertilization (IVF) and development (IVD). COCs were collected from antral follicles of porcine ovaries collected from abattoir, and were maturated in modified NCSU-23 (mNCSU-23) with 10% pFF, 0.6 mM cysteine, 50 ${\mu}mM{\beta}-mercaptoethanol$, 1 mM dbcAMP, 10 IU/mL PMSG and 10 IU/mL hCG, which was supplemented with or without 10 ng/mL EGF and into which 50 or 15 COCs per droplet was put. Oocytes matured in vitro, were fertilized in vitro in modified Tris-buffered medium (mTBM) with the final motile sperm concentration of 1${\times}$105 sperm/mL, and subsequently putative embryos were developed in vitro in NCSU- 23. The results are as follows. 1.In the result of IVM, 10 ng/mL EGF supplement duplicated the percentage of C4 group of COCs(41% vs 81%). But the rate of germinal vesicle breakdown (GVBD) and of nuclear maturation were not significantly different between control and EGF supplemented, or between the number of COCs per culture droplet, and there was not a significant interaction between the two factors, either. 2. In the result of IVF, there was not significantly different between control and EGF supplemented, or between the number of COCs per culture droplet, or was not a significant interaction between the two factors, in the rate of sperm penetration, in the percentage of oocytes with male pronucleus (MPN), and in the rate of polyspermy. 3. In the result of IVD, there was not significantly different between control and EGF supplemented, or between the number of COCs per culture droplet in the percentage of cleaved oocytes. There was not significantly different between the number of COCs per culture droplet, but between control and EGF supplemented (p<0.01) in the percentage of blastocysts, the number of inner cell mass (ICM), trophectoderm (TC) and total cells. There was no significant interaction between the two factors anywhere. These results suggested that 10 ng/mL EGF supplement into mNCSU-23 for IVM was effective in the production of more as well as better blastocysts during IVD through increasing the number of cells in those.

• Journal of Embryo Transfer
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• v.19 no.3
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• pp.245-255
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• 2004

In recent years, an increasing number of studies on pig in vitro maturation(IVM) and in vitro fertilization(IVF) have been separated. the wide range of new technologies, including that in applied molecular genetics, has increased this interest. the production of viable porcine embryos in vitro is a prerequisites for the successful production of transgenic pigs to date. The efficiency of IVM/IVF techniques in the porcine is lower than that obtained in other species such as cattle and mouse. The several problems are generally thought to be the cause of poor results: the low rate of MPN formation derived from inadequate IVM of oocytes, the high incidence of polyspermy after IVF and cell blocking at 4 cell during embryos culture. For there reasons overcoming, many studies have been conducted to improve in vitro embryo-genic competence of oocytes. In the last several years, many maturation culture media have been evaluated and various exogenous factors such as hormones and grows factors have been tested to improve the efficiency of porcine in vitro system. In the study several antioxidants have been examined to improve in vitro fertilization and development of porcine oocytes. In this study, several antioxidants were examined to determine the effects on the development of oocytes to the cleavage, morula and blastocyst stage when added at the maturation(IVM) or in vitro fertilization(IVF) or in vitro culture(IVC) of porcine embryos. Porcine oocytes were matured, fertilized and embryos were cultured in defind conditioned medium in vitro with or without supplementation with the antioxidents of cysteine, catalase and glutathione. 1. Significant improvement of blastocyst rate (27.2％ versus 15.4％, p<0.05) were achieved when catalase(500U/$m\ell$) were added to TCM-199 medium and morula rate(72.0％ versus 53.9％, p<0.05) were significantly higher when glutathione(1.0mM/$m\ell$) were added to TCM-199 medium than those of control. 2. In mTBM medium for oocytes fertilization, the addition of cysteine, catalase and glutathione had no positive effect on embryonic development. glutathione had no positive effect on embryonic development. In conclusion, this study shows that addition of catalase, gluththione during IVM improved the rate of porcine embryo development.

• Journal of Animal Science and Technology
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• v.54 no.4
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• pp.283-290
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• 2012

Cryopreservation induces sublethal damage to the spermatozoa, which leads to their reduced fertile life. The objective of this study was to investigate the effect of taurine, hypotaurine and trehalose as antioxidants on the function of the freezing-thawed sperm in Korean Jeju Black Bull. The semen was cryopreserved with tris egg yolk extendercontaining 7% glycerol and treated with 20mM taurine, hypotaurine and trehalose. Frozen-thawed sperms were evaluated for sperm motility, viability, membrane integrity, acrosome integrity and sperm penetration ability. The results were compared to semen cryopreserved in tris egg yolk extender containing 7% glycerol only as control. Frozen-thawed semen evaluation clearlyindicated that the addition of taurine or hypotaurine significantly improved (p<0.05) the motility and viability compared to control spermatozoa. Moreover, in membrane integrity, swollen sperm ratio was significantly increased (p<0.05) in taurine, hypotaurine or trehalose compared to control. In sperm acrosome integrity, F pattern ratio was increased (p<0.05) in hypotaurine among treatments, and AR pattern was significantly lowered (p<0.05) in taurine, hypotaurine and trehalose. In assessed sperm fertilizing ability, taurine, hypotaurine or trehalose significantly improved (p<0.05) the ratio of pronucleus formation and SFI. Finally, compared with the control, addition of taurine, hypotaurine or trehalose as an antioxidant to the freezing extender showed more positive effects on the frozen-thawed spermatozoa. It is concluded that the addition of taurine, hypotaurine, or trehalose to the freezing extender could reduce cryodamage of the Korean Jeju Black Bull spermatozoa.

• Reproductive and Developmental Biology
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• v.28 no.1
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• pp.21-27
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• 2004

This study was conducted to investigate the developmental ability of caprine embryos after somatic cell interspecies nuclear transfer. Recipient bovine and porcine oocytes were obtained from slaughterhouse and were matured in vitro according to established protocols. Donor cells were obtained from an ear-skin biopsy of a caprine, digested with 0.25% trypsin-EDTA in PBS and primary fibroblast cultures were established in TCM-199 with 10% FBS. The matured oocytes were dipped in D-PBS plus 10% FBS ＋ 7.5 $\mu$ g/ml cytochalasin B and 0.05M sucrose. Enucleation were accomplished by aspirating the first polar body and partial cytoplasm which containing metaphase II chromosomes using a micropipette with an out diameter of 20∼30 $\mu$m. A Single donor cell was individually transferred into the perivitelline space of each enucleated oocyte. The reconstructed oocytes were electric fusion with 0.3M mannitol fusion medium. After the electrofusion, embryos were activated by electric stimulation. Interspecies nuclear transfer embryos with bovine cytoplasts were cultured in TCM-199 medium supplemented with 10% FBS including bovine oviduct epithelial cells for 7∼9 day. And porcine cytoplasts were cultured in NCSU-23 medium supplemented with 10% FBS for 6 ∼8 day at $39^{\circ}C, 5% CO_2 $in air. Interspecies nuclear transfer by recipient bovine oocytes were fused with electric length 1.95 kv/cm and 2.10 kv/cm. There was no significant difference between two electric length in fusion rate(47.7 and 44.6%) and in cleavage rate(41.9 and 54.5%). Using electric length 1.95 kv/cm and 2.10 kv/cm in caprine-porcine NT oocytes, there was also no significant difference between two treatments in fusion rate(51.3 and 46.1%) and in cleavage rate(75.0 and 84.9%). The caprine-bovine NT oocytes fusion rate was lower(P＜0.05) in 1 pulse for 60 $\mu$sec(19.3%), than those from 1 pulse for 30 $\mu$sec(50.8%) and 2 pulse for 30 $\mu$sec(31.0%). The cleavage rate was higher(P＜0.05) in 1 pulse for 30 $\mu$sec(53.3%) and 2 pulse for 30 $\mu$sec(50.0%), than in 1 pulse for 60 $\mu$sec(18.2%). The caprine-porcine NT oocytes fusion rate was 48.1% in 1 pulse for 30 $\mu$sec, 45.2% in 2 pulse for 30 $\mu$sec and 48.6% in 1 pulse for 60 $\mu$sec. The cleavage rate was higher(P＜0.05) in 1 pulse for 30 $\mu$sec(78.4%) and 1 pulse for 60 $\mu$sec(79.4%), than in 2 pulse for 30 $\mu$sec(53.6%). In caprine-bovine NT embryos, the developmental rate of morula and blastocyst stage embryos were 22.6% in interspecies nuclear transfer and 30.6% in parthenotes, which was no significant differed. The developmental rate of morula and blastocyst stage embryos with caprine-porcine NT embryos were lower(P＜0.05) in interspecies nuclear transfer(5.1%) than parthenotes(37.4%).

• Korean Journal of Animal Reproduction
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• v.26 no.1
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• pp.17-23
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• 2002

The present study was carried out to investigate the effects of the maturation media such as a modified TCM-199 (mTCM-199) medium, modified Waymouth MB 752/1 (mWaymouth MB 752/1) medium or NCSU-23 medium on penetrability of pig oocytes by liquid boar sperm. Oocytes (30~40) were transferred into each well of a Nunc 4-well multidish containing 0.5 $m\ell$ maturation medium. When immature pig oocytes were cultured in mTCM-199, mWaymouth MB 752/1 and NCSU-23 maturation media for 44 h in 5% $CO_2$, in air at 38.5$^{\circ}C$, the germinal vesicle breakdown (CVBD) rates of the oocytes were 95.6, 94.1 and 94.9%, respectively, and the maturation rates (metaphase II) of oocytes were 92.5, 90.1 and 91.1%, respectively. No differences were observed among the maturation media. The spermrich portion of ejaculates with greater than 90% motile sperm were used in the experiment. The semen was cooled 22 to 24$^{\circ}C$ over 2 h period. The semen was diluted with Beltsville Thawing Solution (BTS) extender at room temperature to give 2$\times$10$^{8}$ sperm/$m\ell$ in 100 $m\ell$ plastic bottle. Liquid boar semen of 30 $m\ell$ in 100 $m\ell$ plastic bottle was kept at 17$^{\circ}C$ for 5 days. The sperm with greater than 70% motility after day 5 of storage were used for in-vitro fertilization (IVF). After 44 h maturation of immature oocytes, cumulus cells were removed and oocytes (30~40) coincubated far 6 h in 0.5 $m\ell$ mTCM-199 and mTBM fertilization media with 2$\times$1061$m\ell$ sperm concentration. At 6 h after IVF, oocytes were transferred into 0.5 $m\ell$ mTCM-199 and NCSU-23 culture media for further culture 6 or 42 h. Sperm penetration, polyspermy and male pronuclear formation of oocytes at 12 h after IVF, and developmental ability of oocytes at 48 h after IVF were evaluated. The oocytes in combination with NCSU-23 medium for maturation and mTBM medium for IVF increased male pronuclear formation (48.0%) compared to those in combination with mTCM-199 media for maturation and IVF, and mWaymouth MB 752il medium for maturation and mTCM-199 medium far IVF. The rates of cleaved embryos (2~4 cell stage) at 48 h after IVF were 24.1% in combination with mTCM-199 media for maturation, IVF and culture, 43.6% in combination with mWaymouth MB 75211 medium fur maturation and mTCM-199 media for IVF and culture, and 71.2% in combination with NCSU-23 medium for maturation, mTBM medium for IVF and NCSU-23 medium for culture. In conclusion, we found out the oocytes matured in vitro were fertilized by liquid boar sperm stored in BTS extender at 17$^{\circ}C$ for 5 days. We recommend the simple defined NCSU-23 medium for nuclear maturation, mTBM medium and liquid boar sperm for IVF, and NCSU-23 medium for embryo culture.

• Korean Journal of Animal Reproduction
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• v.26 no.1
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• pp.1-7
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• 2002

The present study was carried out to investigate the effects of maturation media on penetrability of pig oocytes by liquid boar sperm coincubated with different sperm concentrations in a modified Tris­buffered medium (mTBM). Follicular oocytes collected from ovaries of prepubertal gilts were matured in a modified TCM-199 (mTCM-199) medium, modified Waymouth MB 752/1 (mWaymouth MB 752/1) medium or NCSU-23 medium. Oocytes (30~40) were transferred into each well of a Nunc 4-well multidish containing 0.5 $m\ell$ maturation medium. The sperm­ich portion of ejaculates with greater than 90% motile sperm were used in the experiment. The semen was cooled 22 to 24$^{\circ}C$ over 2 h period. The semen was diluted with Beltsville Thawing Solution (BTS) extender at room temperature to give 2$\times$10$^{8}$ sperm/$m\ell$ in 100 $m\ell$ plastic bottle. Liquid boar semen of 30 $m\ell$ in 100 $m\ell$ plastic bottle was kept at 17$^{\circ}C$ far 5 days. The sperm with greater than 70% motility after day 5 of storage were used for in-vitro fertilization (IVF). After 44 h maturation of immature oocytes in 5% $CO_2$in air at 38.5$^{\circ}C$, cumulus cells were removed and oocytes (30~40) were coincubated for 6 h in 0.5 $m\ell$ mTBM fertilization medium with five different (1$\times$10$^{6}$ , 2$\times$10$^{6}$ , 4$\times$10$^{6}$ , 6$\times$10$^{6}$, 10$\times$10$^{6}$ $m\ell$) sperm concentrations. At 6 h after IVF, oocytes were transferred into 0.5 $m\ell$ NCSU-23 culture medium fur further culture of 6 h. At 12 h after IVF, sperm penetration, polyspermy and male pronuclear formation of oocytes were evaluated. Oocytes of NCSU-23 maturation medium decreased polyspermy and increased male pronuclear formation compared to those of mTCM­199 and mWaymouth MB 752/1 maturation media. Of oocytes matured in NCSU-23 medium and inseminated in mTBM medium with 2~4$\times$10$^{6}$ $m\ell$ sperm concentrations, 50.8~50.9% showed sperm penetration, 13.3~19.5% polyspermy and 43.9~45.4% male pronuclear formation. In conclusion, we found out that oocytes matured in NCSU­23 medium and inseminated in mTBM medium showed superior in­vitro fertilization compared to those matured in mTCM­199 and mWaymouth MB 752/1 maturation media and inseminated in mTBM medium. The optimum sperm concentrations for in-vitro fertilization of oocytes matured in NCSU-23 medium by liquid boar semen stored at 17$^{\circ}C$ for 5 days were 2~4$\times$10$^{6}$ $m\ell$.