• Clinical and Experimental Reproductive Medicine
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• v.29 no.3
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• pp.179-186
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• 2002

Objective: Lectins are cell-agglutinating and sugar specific proteins or glycoproteins of non-immune origin that precipitate glycoconjugates having saccharides of appropriate complementarity. Because of these properties, plant lectins have been used to help characterize the carbohydrate moieties of glycoproteins in the zona pellucida (ZP) of several mammalian species including pigs. Treatment of oocytes with various lectins blocks sperm binding to the ZP in various mammalian species. This study was undertaken to examine the distribution of sugar residues in the ZP of pig oocytes matured in vitro and the ability of spermatozoa to bind to ZP and in vitro penetration in oocytes treated with fluorescein isothiocyanate (FITC)-labelled lectins. Materials and Methods: The lectins of Banderiaea simplicifolia (BS-II, bind to $\beta$-D-N-acetylglucosamine), Canavalin ensiformis (Con A, bind to $\alpha$-D-Mannose), Lens culinaris (LCA, bind to a-D-Mannose), Ricinus communis (RCA-I, bind to $\beta$-D-Galactose) and Ulex europaeus (UEA-I, bind to $\alpha$-L-Fucose) were examined for spermatozoa penetration, binding capacity to ZP and distribution of lectins. Results: The penetration rates were significantry (p<0.05) higher in control oocytes (63%) than those treated with all lectins, but penetration rates ($40{\sim}49%$) were simililar in group treated with lectins. The incidence of monospermy was similar in oocytes untreated and UEA-I, but it was higher in oocytes treated with BS-II, Con A, RCA-I and LCA. The porcine oocytes cultured for 48 h in TC-199 medium were freed from cumulus cells and treated for 30 min with fluorescein isothiocyanate-labelled lectins. When examined under fluorescein illumination, higher (p<0.001) proportions of oocytes showed fluorescein of zona pellucida after treatment with Con A (93%), LCA (93%) and RCA-I (100%) than BS-II (37%) and UEA-I (50%). All of the oocytes treated with RCA-I exhibited strong fluorescein in the outer region of the zona pellucida while those treated with LCA exhibited strong fluorescein throughout the zona pellucida. BS-II bounded mainly to the outer region and UEA-I bounded mainly to the inner region of the zona pellucida, with either strong or weak fluorescein. At 120 min after insemination in vitro, fewer spermatozoa were bound to the zona pellucida of the oocytes treated with BS-II, Con-A and RCA-I. Of the lectins, Con A most inhibited sperm binding. Conclusions: These results suggest that $\beta$-D-Galactose residues in the porcine zona pellucida may act as primary sperm receptors and inducers of the sperm acrosome reaction and these sugar residues may be involved in the block to polyspermy.

• Asian-Australasian Journal of Animal Sciences
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• v.17 no.2
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• pp.164-167
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• 2004

This study was carried out to investigate in vitro fertilization and development of in vitro matured pig oocytes inseminated with the Duroc boar sperm by different sperm washing media after thawing of the 5 ml frozen straws. Immature follicular oocytes (30-40) were transferred into each well of a Nunc 4-well multidish containing $500{\mu}l$ mTCM199 maturation medium. The sperm rich portion of ejaculates was collected into a 250 ml insulated vacuum bottle and gradually cooled 22 to $24^{\circ}C$ over a 2 h period. Semen was centrifuged at 800 g for 10 min and the seminal plasma discarded. Sperm were esuspended in a lactose-egg yolk and N-acetyl-Dglucosamine (LEN) diluent to contain $1{\times}10^{9}$ sperm/ml and cooled to $5^{\circ}C$ over a 2 h period. Immediately before freezing, semen was rediluted with an equal volume of LEN+4% glycerol and packed into 5 ml straws. After thawing of the 5 ml straw, the 5 ml semen was diluted with 20 ml Beltsville thawing solution (BTS) at room temperature. Oocytes were inseminated with untreated (unwashed and nonpreincubated) or treated sperm (washed two times in BTS, mTLP-PVA and mTBM media, respectively and nonpreincubated) with $2{\times}10^{7}$ sperm concentration. Oocytes were coincubated for 6 h in $500{\mu}l$ mTBM fertilization. At 6 h after IVF, oocytes were transferred into $500{\mu}l$ NCSU-23 culture medium for further culture of 6 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. Sperm penetration rate, male pronuclear formation and rate of cleaved embryos were higher in the BTS, mTLP-PVA and mTBM treatments than the unwashed treatment (p<0.05). The rate of blastocysts from the cleaved oocytes (2-4 cell stage) were higher in the mTLP-PVA treatment than in the unwashed, BTS and mTBM treatments. In conclusion, we recommend the washing of frozen-thawed sperm with mTLP-PVA medium before in vitro fertilization of oocytes in mTBM medium.

• Applied Microscopy
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• v.41 no.2
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• pp.123-128
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• 2011

The micropyles on the prefertilized and artificial fertilized eggs of Epinephelus fasciatus were investigated using scanning electron microscope (SEM). The micropyles ($6.6{\pm}0.41\;{\mu}m$) of E. fasciatus eggs were found in the animal pole and theirs shape were observed as a flat crateriform of cylindrical shape. The micropylar vestibule arranged by 6~7 thickened spiral annuli on the ridge and contributed to differentiate and form fertilization cone for blocking to polyspermy by presenting swollen vestibule structure. As E. fasciatus eggs was pelagic, so chorionic surface was an uneven structures such as circular and fillar form nodules. Especially, various pores (0.15~0.55 ${\mu}m$, 230~270 pores) distributed at the only around micropyles, those pores radiately exhibited regular projection structures showing gill filament-shape. These ultrastructural characters of E. fasciatus eggs can be utilized in a taxonomical cue of grouper species.

• Development and Reproduction
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• v.3 no.1
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• pp.21-28
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• 1999

In order to examine the effect of $Ca^{2+}$ -chelation on in vitro fertilization of rat zona-free oocyte, the formation of cortical granule envelope (CGE) and the rate of fertilization related to monospermy and polyspermy were determined. The ultrastructural characteristics of oocytes were observed with the scanning electron microscope and BAPTA/AM was used for calcium-chelation. The CGE formed by cortical reaction was observed in zona-free oocyte inseminated in vitro and it was also observed in the calcium chelator (1, 5, 10$\mu$M BAPTA/AM) treated zona-free oocytes inseminated in vitro. The CGE developed according to incubation time. The fertilization rate was decreased in the calcium chelator-treated group (59.8, 38.1, 37.0%) compared to the control group (60.6%) but monospermy rate was increased in the calcium chelator-treated group (45.0, 47.3, 50.9%) compared to control group (37.5%). The above results demonstrate that the CGE is formed during fertilization in rat and the extracellular calcium is used in cortical reaction. Also the results suggest that proper concentration of free calcium in oocyte acts as important factor in fertilization.n.

• Journal of Embryo Transfer
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• v.21 no.2
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• pp.129-135
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• 2006

This study was conducted to investigate the effect of in vitro maturation (IVM) duration of porcine follicular oocytes on maturation rate, polyspermic rate, and subsequent embryo development. The nuclear maturation rates of oocytes matured for 36, 38, 40, 42 and 44 hr were similar between 68.0, 78.0, 79.5, 73.8 and 81.8% respectively. There was no significant difference in the rates of polyspermy after in vitro feritilization (IVF). The cleavage rate in the group of 36 hr was significantly higher in than that of 40, 44 hr (p<0.05) but not to 38 and 42 hr. The development rate to blastocyst stage was significantly higher in the group of 38 hr (23.1%) than that in the group of 44 hr (15.6%) (p<0.05) but not to 36, 40 and 42 hr. These results suggest that the aged oocytes for 44 hr is not required for the production of bias to cysts derived from porcine IVF embryos.

• Korean Journal of Animal Reproduction
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• v.15 no.1
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• pp.41-47
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• 1991

The ability of fertilization in vitro and subsequent development of superovulated oocytes was assessed in a controlled environment using an in vitro fertilization technique. The in vitro fertilization percentage of oocytes with cumulus mass declined significantly(P<0.05) with increased doses from 4~10 to 16~40IU of PMSG for superovulation. However, the porportion of polyspermic penetration varied from 2.3 to 9.7% and there was no significant difference between treatments in incidence of polyspermy. When morphologically normal ova with cumulus mass were cultured for 66~72h in a plastic mini-straw to undergo fertilization in vitro, the mean percentage of embryos developed to 2-16 and 4-16cell stage was 61.8 and 17.6%; it was slightly(P<0.05) superior to the corresponding results from petri dish. A total of 52 two-cell embryos fertilized in a mini-straw were transferred to seven pseudopregnant rat. Among these recipients, two normal young were born from one recipient which received a total of six embryos. These results suggest that superovulated oocytes are proportionately less competent than normally ovulated oocytes to undergo fertilization in a controlled environment using an in vitro fertilization technique. Also, a plastic mini-straw designed was slightly superior to petri dish as a culture vessel for fertilization and embryo development in vitro.

• Journal of The Korean Society of Grassland and Forage Science
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• v.38 no.4
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• pp.320-324
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• 2018

In this study, we examined effect of sperm penetration of oocytes after in vitro fertilization (IVF) with cauda epididymal spermatozoa in Hanwoo bull after feeding of timothy hay. One testicle with epididymides was castrated from one Hanwoo bull (14 months of age) and spermatozoa recovered from cauda epididymis and cryopreserved. As control, frozen Hanwoo semen was used. Matured cumulus oocyte complexes were co-incubated with frozen-thawed cauda epididymal spermatozoa for 12 or 18 hours. After IVF, presumptive zygotes were cultured in modified synthetic oviductal fluid. In experiment 1, we examined sperm penetration rate at 12 hours of IVF with epididymal sperm. Total penetration rate among cauda epididymis and control was similar(mean${\pm}$standard error, cauda epididymis and control vs. $49.7{\pm}11.3$ and $54.4{\pm}12.8%$). In experiment 2, cleavage and blastocyst developmental rate were evaluated at day 2 and day 8 after IVF for 18 hours. Cleavage rate among cauda epididymis and control was similar(cauda epididymis and control vs. $81.2{\pm}3.4$ and $82.7{\pm}2.5%$). However, blastocyst developmental rate of cauda epididymis group was significantly higher than that of control group(cauda epididymis and control vs. $24.4{\pm}1.6$ and $12.2{\pm}2.8%$, p<0.05). In conclusion, cauda epididymal spermatozoa in Hanwoo bull has high embryo developmental competence and can be used as an alternative to ejaculated frozen sperm in vitro.

• Proceedings of the KSAR Conference
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• 2003.06a
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• pp.63-63
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• 2003

This study was carried out to investigate the effects of liquid boar sperm stored at 4$^{\circ}C$ on sperm motility, normal acrosome, and in-vitro fertilization and culture of pig oocytes matured in-vitro. The sperm-rich fraction (30~60 ml) of ejaculate was collected into an insulated vacuum bottle. Semen was slowly cooled to room temperature (20~23$^{\circ}C$) by 2 h after collection. Semen was transferred into 15 ml tubes, centrifuged at room temperature for 10 min at 800$\times$g, and the supernatant solution was poured off. The concentrated sperm was resuspended with 5 ml of lactose, egg yolk and N-acetyl-D-glucosamine (LEN) diluent to provide 1.0$\times$10$^{9}$ sperm/ml at room temperature. The resuspended semen was cooled in a refrigerator to 4$^{\circ}C$ and preserved for 5 days to examine sperm motility and normal acrosome. The medium used for oocyte maturation was modified tissue culture medium (TCM) 199. After about 22 h of culture, oocytes were cultured without cysteamine and hormones for 22 h at 38.5$^{\circ}C$, 5% $CO_2$ in air. Oocytes were inseminated with liquid boar sperm stored at 4$^{\circ}C$ for 2 days after collection. Oocytes were coincubated for 6 h in 500 ${mu}ell$ mTBM fertilization media with 0.2, 1, 5 and 10$\times$10$^{6}$ /ml sperm concentration, respectively. At 6 h after IVF, oocytes were transferred into 500 ${mu}ell$ Hepes-buffered NCSU-23 culture medium for further culture of 6, 48 and 144 h. There were significant differences in sperm motility and normal acrosome among preservation days and incubation times, respectively. The rates of sperm penetration and polyspermy were higher in 5 and 10$\times$10$^{6}$ sperm/ml than in 0.2 and 1$\times$10$^{6}$ sperm/ml. Male pronuclear formation was lower in 0.2$\times$10$^{6}$ sperm/ml than in 1, 5 and 10$\times$10$^{6}$ sperm/ml. Mean numbers of sperm in penetrated oocyte were highest in 10$\times$10$^{6}$ sperm/ml compared with other sperm concentrations. The rate of blastocysts from the cleaved oocytes (2~4 cell stage) was highest in 1$\times$10$^{6}$ sperm/ml compared with other sperm concentrations. In conclusion, we found out that liquid boar sperm stored at 4$^{\circ}C$ could be used for in-vitro fertilization of pig oocytes matured in-vitro. Also, we recommend 1$\times$10$^{6}$ ml sperm concentration for in-vitro fertilization of pig oocytes.

• 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.

• Reproductive and Developmental Biology
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• v.29 no.3
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• pp.201-205
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• 2005

The objective of this study was to investigative the effects of amino acids supplementation on maturation, fertilization and embryo development of pig oocytes. Essential amino acids (EA), non-essential amino acids (NA) or both amino acids (EA + NA) were supple-mented to North Carolina State University (NCSU) 23 medium containing porcine follicular fluid (pFF). When the amino acids were supplemented to the maturation medium, the maturation rates were higher (p<0.05) in the NA group than control ($83.3{\pm}0.04\%\;versus\;70.0{\pm}0.05\%$, but the subsequent cleavage rates and development to morula and blstocyst stage between aminoacid supplement groups and control were not different. The developmental rates to morula and blastocysts stage were not significantly different regardless of amino acid supplementation to culture medium. In addition, supplementation of amino acids did not significantly affect the rate of fertilization and polyspermy. When the amino acids were supplement to culture medium, the number of trophectodermal (TE) cells was significantly (p<0.05) higher in amino acid supplement group than that of control ($18.6{\pm}0.5\;versus\;16.1{\pm}0.6$), whereas the numbers of inner cell mass (ICM) cells were not different among the treaonent groups and control ($29.0{\pm}0.9\~31.5{\pm}1.2$). Total cell number was also significantly (p<0.05) higher in EANA group ($50.0{\pm}1.0$) than that of control group ($44.2{\pm}1.1$). These results indicate that the amino acid supplementation to maturation and culture medium may not significantly stimulate early embryo development, but may improve the TE cell number of blastocyst stage in the pig.