• Reproductive and Developmental Biology
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• v.34 no.1
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• pp.21-25
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• 2010

The purpose of this study was to investigate the effects of the collection time, co-culture and sperm penetration of canine oocytes on in vitro maturation and fertilization. The oocytes were cultured in TCM-199 media containing hormonal supplements (10% FCS, 10 IU/ml HCG, 10 IU/ml PMSG) at 5% $CO_2$, 95% air, $38^{\circ}C$. The in vitro maturation rate to MII stage of in vitro oocytes recovered from ovaries that collected at follicular, luteal and inactive phases of the reproductive phase for 44~72 hrs were 19.2%, 12.2%, and 6.0%, respectively. Follicular phases oocytes had a significantly higher in vitro maturation rate than oocytes collected at luteal and anestrus stage (p<0.05). The in vitro maturation rates to the MII stage of canine oocytes after 48 hrs of culture with glutathione, pyruvate, or glutathione + pyruvate were 12.5%, 10.7%, and 17.5%, respectively. This was higher than that in both alone or the combination of the two compared to the control group (19.0%). The sperm penetration rates of in vitro matured oocytes by fresh and frozen semen were 29/80 (36.3%) and 18/80 (22.5%), respectively. Although there are limited reports about canine oocytes co-culture and in vitro fertilization, our results on in vitro maturation is comparable to the results from other researches.

• Asian-Australasian Journal of Animal Sciences
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• v.15 no.2
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• pp.172-178
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• 2002

The present study examined whether treatment of in vitro matured pig oocytes with calcium ionophore (A23187) could prevent polyspermic penetration in vitro. When oocytes cultured for maturation for 33, 36 or 44 h were subsequently treated with $50{\mu}M$ A23187 in medium with fetal calf serum (FCS) for 1, 2 and 3 h and then cultured for 12 h without spermatozoa, virtually no activation occurred. In the absence of FCS, however, 31-42, 45-49 and 56-64% of oocytes were activated, respectively. When oocytes treated with $50 {\mu}M$ A23187 in medium with FCS for 3 h were inseminated in vitro, the penetration rates (14-57%) were lower (p<0.01) with a higher (p<0.01) incidence (35-67%) of monospermy compared with untreated oocytes (69-80% penetration and 15-17% monospermy). However, sperm penetration was completely blocked in all oocytes treated with A23187 in the absence of FCS. When oocytes matured for 33 h were treated with different concentrations of A23187 for 3 h and inseminated in vitro, the penetration rate did not change but there was an increased incidence (p<0.05) of monospermy at $10-20{\mu}M$ and $2.5-5{\mu}M$ A23187 in the presence and absence of FCS, respectively, compared with at $0{\mu}M$ A23187. With these lower concentrations of A23187, treatment of oocytes for at least 60 and 30 min in the presence and absence of FCS, respectively, was required to increase the incidence of monospermy without reducing penetration rate. These results indicate that a high concentration ($50{\mu}M$) of A23187 in medium without FCS, but not in medium with FCS, stimulated in vitro matured pig oocytes to induce parthenogenetic activation and a complete block to sperm penetration in vitro. However, treatment of oocytes with lower concentrations of A23187 ( $10-20{\mu}M$ and $2.5-5{\mu}M$) both in the presence and absence of FCS maintained sperm penetration in vitro and increased the incidence of monospermy.

• Journal of the Society of Cosmetic Scientists of Korea
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• v.40 no.1
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• pp.1-10
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• 2014

At present, there are few research papers on skin penetration of cosmeceutical ingredients. What is worse is that in vivo studies are hard to find. In this study, we measured skin epidermal penetration of cosmeceutical ingredients using in vivo Raman spectroscopy and compared with the results obtained from experiments using in vitro franz cell. Results showed that ascorbyl-2-glucoside, retinol, retinyl palmitate, and kojic acid were good for penetration ratio in measurement in vitro and retinol, vitamin C, and arbutin were good in measurement in vivo. Among them, retinol was best in skin penetration in vivo experiment using Raman spectroscopy and ascorbyl-2-glucoside was best in skin penetration in vitro experiment using Franz cell system. It is estimated that the differences were originated from the experimental procedures of two different methods; in vivo Raman experiment can be sensitive to the effect of epidermis and dermis as characteristics of matter by estimating the stratum corneum and in vitro measurement is evaluation of material to penetrate skin of hairless mouse. However, most penetration barrier is the stratum corneum, thus it is important to examine movement of material in the stratum corneum. We expect that these results provided useful information for many cosmetic related research.

• Journal of Embryo Transfer
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• v.10 no.3
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• pp.237-242
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• 1995

Porcine follicular oocytes matured in culture were inseminated with frozen-thawed spermatozoa. When the oocytes were inseminated in the medium with oviductal epithelial cell monolayer, the penetration rates higher in those with (4.1, 31.7, 45.1, 54.5 and 69.4%) than without cells (0, 17.1, 34.8, 45.2 and 58.9%) at 4, 8, 12, 16 and 20 h after insemination. The proportions of polyspermy in penetrated oocytes in medium with or without cells increased with time of examine. In another experiment, the penetration rate was higher without (57.6%) than with (19.6~24.1%) preincubation of spermatozoa for 1~4 h in medium. However, when the oocytes were inseminated with spermatozoa preincubated for 1~2 h, the penetration rates significantly higher (P<0.05) in those with (65.6 and 55.9% for 1 and 2 h) than without (24.1 and 20.6% for 1 and 2 h) oviductal epithelial cell monolayer. On the other hand, the proportions of polyspermy decreased with time of spermatozoa preincubation. These results indicate the significant advantages of the spermatozoa preincubation with oviductal epithelial cell monolayer for 1 and 2 h to maintain penetration potential during in vitro fertilization in the porcine.

• Korean Journal of Veterinary Service
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• v.26 no.4
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• pp.329-337
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• 2003

The functional role of the cumulus cells on sperm penetration and polyspermy during in vitro fertilization was examined. The penetration rate was significantly higher(p<0.01) in oocytes with(61%) than without(25%) cumulus cells. No significant differences, however, was observed in polyspermy. When the hyaluronidase was supplemented to the fertilization medium with different concentrations, penetration rates in oocytes with cumulus cells were higher than oocytes without cumulus cells at 0(61 vs 34% ; p<0.05), 0.01(56 vs 35% ; p<0.05), 0.1(66 vs 30% ; p<0.05) and 1.0 mg/$m\ell$(39 vs 27%). On the other hand, the polyspermy rates were lower oocytes without than with cumulus cells, and had a tendency to decrease with high concentrations of hyaluronidase. In another experiment, the penetration and polyspermy rates had a tendency to increase as time of sperm-oocytes culture was prolonged. At 16 and 20hrs after insemination, the penetration rates were significantly higher(p<0.05) in oocytes with(48 and 62% for 16 and 20hrs) than without(25 and 31% for 16 and 20hrs) cumulus cells in medium with hyaluronidase. However, the polyspermy rates were significantly(p<0.05) lower in oocytes without(3 and 16%) than with(37 and 48%) cumulus cells at 16 and 20hrs after insemination. In cumulus-free oocytes inseminated in medium with or without hyaluronidase at different concentrations of cumulus cells, the penetration rates were significantly(p<0.05) higher in medium with than without hyaluronidase at different concentrations of cumulus cells. The proportions of polyspermy were lower in medium without than with hyaluronidase at 0 (10 vs 0%), 10$^2$(25 vs 0%), 10$^4$(24 vs 14%) and 10$\^$6/(29 vs 10% ; p<0.05) cumulus cells/ml. These results suggest the advantage of culture in medium with cumulus cells and denuded oocytes to inhibit polyspermy with no decrease in the penetration rates during the fertilization in vitro in the porcine.

• Restorative Dentistry and Endodontics
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• v.21 no.1
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• pp.242-253
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• 1996

Penetration degree of several dyes into dentinal tubules in vivo was compared with in vitro, and experimental sensitivity of those dyes was investigated in 64 canines of 16 cats. Dentin 1mm below cusp tip was exposed by cross-sectioning with diamond disc. Pulp of 32 canines were extirpated through cervical cavities. In the presence of smear layer or after add-etching with 37% phosphoric acid to the exposed dentin surface, acrylic collar and resin cap was attached to keep dyes. 52 mmol/$\ell$ Evans' blue, 2% Methylene blue, 10 % Silver nitrate and 5% Fluorescene were then applicated on each 4 canines respectively for 30 minutes. After rinsing, the canines were ground-sectioned longitudinally and linear dye penetration was measured under microscope. The results obtained were as follows ; 1. Evans' blue and Methylene blue penetrated significantly(P<0.05) more in vitro than in vivo only in the cases that exposed dentin surfaces were not etched with acid. 2. Silver nitrate penetrated significantly(P<0.05) more in vitro than in vivo in both cases that exposed dentin surfaces remained intact and were etched with acid. 3. The penetration degree of Fluorescene did not show statistical significance between in vivo and in vitro or in acid-etched and in not acid-etched cases.

• Toxicological Research
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• v.17 no.1
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• pp.7-9
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• 2001

The quantitative assessment of the penetration of organic solvents through skin is necessary for the evaluation of health hazards in occupational environments. We investigated the rate of dermal penetration when mixed or single forms of organic solvents were placed into a diffusion cell in vitro or into an experimental animal in vivo. The diffusion rates of methanol. toluene, and styrene were 6.07, 0.129, and 0.046 mg/$cm^2$/h, respectively. When skin was exposed to the mixed solvent of methanol and toluene, the penetration rate of toluene did not change significantly (0.110 mg/$cm^2$/h). However, the rate of methanol penetration increased to 43.90 mg/$\textrm{cm}^2$/h. The penetration rate of methanol also increased significantly to 54.69 mg/$cm^2$/h by mixing it with styrene. The concentration of methanol in the blood was monitored during the epicutaneous exposure in rats. The blood concentration of methanol was increased by mixing methanol with toluene as seen in the in vitro experiments. These results showed that the penetration rate of organic solvents would be enhanced by mixing them with other solvents.

• Reproductive and Developmental Biology
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• v.32 no.4
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• pp.237-243
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• 2008

The objective of this study was to examine the effect of caffeine and sodium bicarbonate in a fertilization medium on the fertilizability of boar spermatozoa that were frozen in straws. Boar spermatozoa were extended with Beltsville F5 extender and frozen in 0.25-ml straws. In vitro matured porcine oocytes were fertilized in vitro (IVF) with frozen-thawed boar spermatozoa for 6h in a modified tris-buffered medium (mTBM) or in its modified medium by substituting the tris with 25mM sodium bicarbonate (modified bicarbonate-buffered medium; mBBM). Some of inseminated oocytes were fixed and stained for examination of sperm penetration. IVF embryos were cultured in a North Carolina State University-23 medium for embryo development. The percentage of live sperm was $47{\pm}4%$ and morphological abnormality of acrosome was found in $14{\pm}3%$ of spermatozoa. Optimal sperm concentration for IVF was $0.75{\sim}1.0{\times}1.0{\times}10^6$ sperms/ml when mTBM containing 5mM caffeine was used as the fertilization medium. Sperm penetration was significantly (p<0.05) stimulated by increasing caffeine concentration in the IVF medium. In addition, mBBM significantly (p<0.05) increased sperm penetration (92%) compared to mTBM (65%). More (p<0.05) blastocysts (22% vs. 32%) developed from the oocytes that were fertilized in mBBM containing 1mM caffeine than from those fertilized in mTBM with 5mM caffeine. Our results indicate that boar spermatozoa can be frozen successfully in straws with holding their normal fertilizability and that caffeine and sodium bicarbonate stimulates sperm penetration in vitro.

• Proceedings of the KSAR Conference
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• 2001.03a
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• pp.43-43
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• 2001

This study evaluated the effect of fertilization-promoting peptide (FPP) on fertilizing ability and glycosidase activity in vitro of spermatozoa frozen-thawed in pig, Using chlortetracycline fluorescence analysis, the various glycosidase analyses and the oocyte penetration test, we have obtained evidence that FPP can promote the fertilizing ability and glycosidase activity of frozen-thawed spermatozoa in vitro. When frozen-thawed spermatozoa was washed with different concentrations of FPP, there were significantly (P<0.05) more acrosome-reacted in medium with 100 nM than 0, 50, 200 and 400 nM. The penetration rates were also highest in medium containing with 100 nM FPP (P<0.05). On the other hand, the $\beta$-N-acetylglucosaminidase activity was at least twofold higher than other glycosidase. In same glycosidase, however, there were no difference in medium with different concentrations of FPP In another experiment, spermatozoa preincubated in medium with or without FPP for 0, 1, 2, 3 and 4 h were inseminated with oocytes matured in vitro. The percentages of spermatozoa that reached acrosome reaction were affected by preincubation and were higher in medium with that than without FPP. When oocytes were inseminated with spermatozoa preincubated in medium with and without FPP during the different periods, however, penetration rates were decreased with preincubation periods of spermatozoa. On the other hand, when the sperm-oocyte were cultured for 4, 8, 12, 16, 20 and 24 h, the penetration rates were higher in spermatozoa preincubated with that than without FPP and had a tendency to increase as time of culture periods. However, The activities of $\alpha$-fucosidase, $\alpha$ -mannosidase, $\beta$-galactosidase and N-acetyl- $\beta$-D-glucosaminidase were higher in medium with that than without FPP regardless of periods of sperm preincubation and sperm-oocyte culture. These results suggest that FPP may play a positive role in promoting of sperm function and glycosidase activity in vitro in pig.

• Journal of Embryo Transfer
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• v.14 no.1
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• pp.9-15
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• 1999

This study was conducted to investigate the effect of $\alpha$-tocopherol and cysteamine with Whitten's medium in supporting the development on in vitro maturation(IVM), in vitro fertilization (IVF) and in culture(IVC) on porcine oocytes. When the immature oocytes were cultured of $\alpha$-tocopherol for 40h, the nuclear maturation rates were 39, 4, 52.5 and 54.1%, respectivley. The nuclear maturation rates of treat groups were signficantly (P<0.05) higher than those of non-treat groups. After matureation, the oocytes were inseminated in vitro in medium 199 with ejaculated spermatoza for examination of sperm penetration, polyspermy, male pronuclear(MPN) formation, and cleavage rate. Sperm penetration rates of treat higher than the control groups(P<0.05), and MPN formation rates were significantly(P<0.05) higher on treated groups (24.3~53.1%) than control groups(14.2~21.4%). After insemination, the cleavage rates at 120hr were groups higher than control groups(P<0.05).