• Korean Journal of Animal Reproduction
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• v.16 no.3
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• pp.199-208
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• 1992

These studies were carried out to investigate optimal physological conditions for in vitro fertilization (IVF) of mouse ova. The unfertilized ova were obtained by superovulation from ICR mice of 4 to 6 weeks old. Tyrode's 280 solution was used as basal media, and pH and osmolality of basal media were adjusted with the supplementation of sodium bicarbonate and sodium chloride, respectively. The optimal pH, and osmolality of culture media and the optimum period of sperm preincubation were examined in fertilization in vitro of mouse ova and the subsequent culture in vitro of embryos. The pH range of media examined was designed from 6.5 to 7.5 with 0.2 interval and the range of osmolality from 250 to 370 mOsm with 20 interval, and the period of sperm preincubation examined was 30, 60, 120, and 180 minutes. The ova developed to 2-cell embryosafter 26hrs. of incubation with preincubated sperm were evaluated as in vitro fertilized ones. The results obtained were summarized as follows: 1. The percentage of in vitro fertilized ova was highest (64.7%) in media of pH 7.1 and lowest (38.0%) in pH 6.7. No significant difference in % fertilized ova was found from the media of pH 7.1 to 7.5. Compared with the result from pH 7.1 medium, the pollyspermy was increased signifciantly (p<0.05) in the media of pH over 7.5 and below 6.9;, and the % degenerated ova was significantly (p<0.05) increased in the media of pH below 6.9. 2. The percentage of in vitro fertilized ova was highest (69.4%) in media of osmolality 330 mOsm and lowest (47.9%) in osmolality 250 mOsm. No significant difference in % fertilized ova was found from the media of osmolality 310 to 350 mOsm. Compared with the result from osmolality 330 mOsm in medium, the polyspermy aws increased significantly(p<0.05) in the media of osmolality over 350 mosmol and blow 290 mOsm, and the % degenerated ova was significantly (P<0.05) increased in the media of osmolality below 290 mOsm. 3. The percentate of in vitro fertlilized ova was highest (62.7%) in media of period sperm preincubation 180 min. and lowest (40.4%) in sperm preincubation 30 minutes. No significant difference in % fertilized ova was found from the media of sperm preincubation 120 to 180 minutes. Compared with the result from sperm preincubation 180 minutes in medium, the polyspermy was low differ no significantly(P<0.05) in the media of period sperm preincubation, and the % degenerated ova was signifciantly(P<0.05) increased in the media of sperm presincubation below 60 minutes.

• Journal of Embryo Transfer
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• v.11 no.3
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• pp.225-231
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• 1996

This study was conducted to investigate the effects of preincubation time, concentration and exposure time of sperm on in vitro fertilization of porcine follicular oocytes rnatured in in vitro. The results obtained are as follows ; 1. Effect of preincuhation time for porcine sperm capacitation on in vitro fertilization in medium with heparin was investigated. Normal fertilization rate was highest in 15 min(26.4%). However, there were no significant differences among preincuhation times of 5~90 min, 2. Normal fertilization rates of sperm concentrations were 17.0~26.5%, and normal fertilization rate from l$\times$ l05cell /ml concentration was also higher than those of other sperm concentration. 3. Normal fertilization rates of sperm exposure time of 4, 8, 12, 16 and 20 hours were 6.1, 20.8, 27.8, 25.0 and 26.7%, respectively. Normal fertilization rate from sperm exposure time of 12 hours was also higher than that of other sperm exposure times.

• Korean Journal of Animal Reproduction
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• v.16 no.3
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• pp.209-215
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• 1992

These studies were carried out to overcome 2-cell block and in vitro development to blastocysts in vitro fertilization of mouse embryos. The unfertilized ova were obtained by superovulation in ICR mice of 4 to 6 weeks old. Tyrode's 280 solution was used as basal media, and the pH range of media examined was designed from 6.5 to 7.5 with 0.2 interval and the range of osmolality from 250 to 370 mOsm with 20 interval, and the period of sperm preincubation examined was 30, 60, 120, and 180 minutes. The ova developed to 2-cell embryos after 26hrs of incubation with preincubated sperm were evaluatated as in vitro fertilized ones. The results obtained were summarized as follows: 1. The optimal ranges of pH and osmolality of culture media and of sperm preincubation time for in vitro development of in vitro fertilized ova to blastocyst were pH 7.1 to 7.3, 250 to 350 mosmol and 60 to 180 min, respectively. 2. With the media of pH 7.1, 310 mOsm and sperm preincubation period of 120min in another experiment of large sample size, the in vitro fertilized ova was found 66.5% and the in vitro development of in vitro fertilized ova to blastocyst was found 35.8%. From the above results it was concluded that the optimal conditions of pH and osmolality of the media for mouse IVF and embryo culture, and the period of sperm preincubation might be 7.1, 310 mOam and 120min, respectively.

• Korean Journal of Animal Reproduction
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• v.10 no.1
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• pp.109-120
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• 1986

This study was conducted to define the effect of addition of lysolecithin (LC) and 20% v/v rabbit serum to sperm preincubation medium on the induction of acrosome reaction (AR) an fertilizing ability in vitro of LG-added sperm. Ejaculated rabbit sperm from New Zealand White buck was washed once by centrifugation, then preincubated for 2 or 4 hrs in a chemically defined medium (DM), DM plus 20% rabbit serum or BSA-free DM plus 20% rabbit serum at 37$^{\circ}C$ water bath or CO2 incubator. At the end of preincubation LC was added to the preincubated sperm, which was stained at 0.5 to 4 hr later and examined for AR and sperm motility. For in vitro fertilization, gametes were coincubated in DM up to 24 hrs and thereafter fertilized embryos were incubated in BSM -II up to 48 hrs. Addition of LC to 4-hr preincubated sperm was more effective for the AR and sperm motility than that to 2-hr preincubated sperm and optimal concentration of LC for AR was about 80${\mu}$g/ml. A significant increase in AR occured from 20 to 30 min. after addition of 80 to 100${\mu}$g/ml in 4-hr preincubated sperm. BSA-free DM plus 20% rabbit serum showed a higher AR and sperm motility than those of DM plus 20% rabbit serum in LC-added sperm after 4-hr preincubation. The incidence of AR after 4-hr preincubation and at 30 min after 60${\mu}$g/ml LC addition varied greatly among individual bucks. Sixty ${\mu}$g/ml LC-added sperm showed a slight high cleavage rate over control levels, but 100${\mu}$g/ml LC-added sperm showed lower cleavage rate rather than 60${\mu}$g/ml LC. It is concluded that optimal concentration of LC for high AR induction and sperm motility in 4-hr preincubated sperm was about 80${\mu}$g/ml, but 60${\mu}$g/ml level was more useful for in vitro fertilization.

• Journal of Embryo Transfer
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• v.7 no.2
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• pp.73-79
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• 1992

Effects of caffeine, heparin and caffeine-heparin treatments for in vitro capacitation of Korean Native Cattle sperm on acrosorne reaction and viability were studied using the methods of Wells-Awa and Dual stain. The results were summerized as follows: 1. The acrosome reaction of sperm when treated with caffeine after 0 to 4 hrs of preincubation were 11.0~75.7% for Wells-Awa stain, and 14.3~75.55% for Dual stain. True acrosome reaction of sperm for Dual stain was 3.0~29.2%. The viability of sperm was 62. 2~27.2%. 2. The acrosome reaction of sperm when treated with heparin after 0 to 4 hrs of preincubation were 17.0~81.2% for Wells-Awa, and 14.3~75.5% for Dual Stain. True acrosome reaction of sperm for Dual stain was 1.5~26.6%. The viability of sperm was 58.6~35. 8%. 3. The acrosome reaction of sperm when treated with caffeine-heparin after 0 to 4 hrs of preincubation were 13.0~83.2% for Wells Awa, and 11.0~78.5% for Dual stain. True acrosome reaction of for Dual stain was 5.1~26.3%. The viability of sperm was 60.5~30.1%.

• Korean Journal of Animal Reproduction
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• v.10 no.2
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• pp.192-203
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• 1986

Two experiments in this study were designed to compare the potential for in vitro capacitation and in vitro fertilization of ejaculated sperm among individual rabbit bucks. In experiment 1, for in vitro capacitation, the ejaculated sperm were preincubated in DM for 12 hr or 18 hr after HIS treatment, then 12 hr -or 18 hr- preincubated sperm were incubated with superovulated rabbit ova in a 5% CO2 incubator for 36 hr at 38$^{\circ}C$, and a part of cleaved ova was transferred to the recipient does for implantation of embryo. In experiment 2, effect of lysolecithin addition to preincubation medium on induction of accelerated in vitro capacitation and in vitro fertilization of individual rabbit sperm was studied. Experiment 1; 1. Percent acrosome reaction of sperm, noted after staining, after 12 hr or 18 hr preincubation ranged from 52.5 to 76.0% and from 67.5 to 90.0%, respectively and sperm motility index of these sperm ranged from 20.0 to 47.5 for 12 hr-preincubated sperm and from 15.0 to 37.5 for 18 hr- preincubated sperm. There was no a certain relation between percent acrosome reaction and sperm motility index. 2. In vitro fertilization rate (cleavage rate) of in vitro capacitated sperm varied widely among individual bucks, ranging from 0 to 47.8% for 12 hr - preincubated sperm and from 0 to 60.9% for 18 hr -prein- cubated sperm. Cleavage rate of 18 hr - preincubated sperm was higher and faster than that of 12 hr - preincubated sperm. 3. Eight of 44 in vitro fertilized embryos transferred into 6 recipients were implanted in 4 recipients (66.7%) up to day 15 and implnatation rate was 18.2%. Experiment 2; 1. The percent acrosome reaction of sperm before and after 4 hr preincubation in DM without lysolecithin varied significantly among individual bucks, ranging from 0.4 to 18.4% and from 1.7 to 37.4%, respectively and percent acrosome reaction of sperm at 30 min after addition of 60${\mu}$g/ml lysolecithin also was significantly different among bucks, ranging from 19.2 to 67.1%. 2. Effect of accelerated acrosome reaction following lysolecithin addition was more considerable in the individuals showed less percent acrosome reaction before and after 4 hr preincubation. Percentage of motile sperm and motility score showed a trendency towards a decrease with increase of preincubation time and time after lysolecithin addition. 3. In vitro fertilization rate (cleavage rate) at 24 hr postinesmination with pooled sperm were treated to 60 $\mu\textrm{g}$/ml lysolecithin for 30 min after 4 hr preincubation was 24.6%, a higher rate than 13.2% for control. While 80 $\mu\textrm{g}$/ml lysolecithin-added sperm showed a lower cleavage than control and 60$\mu\textrm{g}$/ml-added sperm at both 24 hr and 48 hr postinsemination. These results from 2 experiments suggest that more useful preincubation time for the in vitro capacitation of ejaculated rabbit sperm is 18 hr in DM after HIS treatment, although there is wide variation in vitro capacitation and in vitro fertilization rate among individual bucks, and lysolecithin addition to at least 4 hr - preincubated sperm in DM can result in almost same in vitro fertilization rate as that of 18 hr - preincubated sperm in the experiment 1.

• Journal of Embryo Transfer
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• v.28 no.1
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• pp.41-48
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• 2013

This study examined the motility of either the unattached(upper) or attached(lower) Hanwoo sperm to bovine oviduct epithelial cell(BOEC) monolayers to determine whether there are any changes in their motility during co-culture. The cleavage and blastocyst development rate were compared among different preincubation methods in-vitro, after oocytes were fertilized in-vitro with Hanwoo sperm on BOEC monolayers. The motility of frozen-thawed sperm in BOEC co-culture group was significantly higher than controls, especially at 5 hours and 6 hours (p<0.05) of incubation, in sperm treatment medium without heparin and caffeine. The motility of frozen-thawed sperm in BOEC co-culture group was significantly higher than controls, especially at 3 hours (p<0.05) and 6 hours (p<0.01), in sperm treatment medium containing heparin and caffeine. The motility of the attached( lower) sperm was significantly higher than the unattached(upper) sperm during co-culture with BOEC at all times(p<0.01 or p<0.05), except for 6 hours. After Hanwoo oocytes were fertilized in-vitro with the sperm that had been co-cultured with BOEC in sperm treatment medium containing heparin and caffeine, we determined the cleavage and blastocyst development rate, according to the preincubation methods. Both the cleavage and blastocyst development rate from 2 hour preincubation group were the highest, but significant difference was not recognized. These results show that BOEC plays an important role on sperm hyperactivation related to capacitation regardless of heparin and caffeine in sperm treatment medium. However, oviduct epithelial cell had no significant effect on the development of embryos after in-vitro fertilization in the presence of added heparin and caffeine in sperm treatment medium.

• Clinical and Experimental Reproductive Medicine
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• v.24 no.1
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• pp.27-34
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• 1997

This study aimed to evaluate the effect of seminal vesicle fluid (SVF) on the acrosome reaction (AR) occurred spontaneously or induced by $Ca^{2+}$ ionophore A23187, follicular fluid, and progesterone in mouse epididymal sperm. SVF was divided into high (MW>10 kD) and low (MW<10 kD) fractions by ultrafiltration. The low MW fraction of SVF decreased the rate of spontaneous AR, however the high MW fraction did not. It suggested that the low MW fraction of SVF might have contained decapacitation factor(s) responsible for prolonging of time need for capacitation. When sperm preincubated for 60 min in the presence of SVF, the rate of AR induced by A23187 was decreased, but prolongation of preincubation time for 120 min significantly potentiated the AR by A23187. It suggested that addition of SVF into sperm preincubation medium imposed the epididymal sperm a condition similar to ejaculation. AR induced by human follicular fluid or progesterone was also inhibited by SVF. It suggested that substance in SVF might have affected AR of mouse sperm by inhibiting the interaction between AR inducing ligands and sperm surface receptors involved in acrosomal exocytosis.

• Korean Journal of Animal Reproduction
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• v.23 no.3
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• pp.229-237
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• 1999

The influence of ascorbic acid (Asc) and ferrous sulfate (Fe$^{2＋}$) on capacitation, acrosome reaction and fertility in vitro was investigated in boar frozen-thawed spermatozoa with or without preincubation. The addition of 0-1.0 mM Fe$^{2＋}$ to sperm suspensions during preincubation increase acrosome reaction (P＜0.05) and oocyte penetration. These increase are also associated with addition of 0~0.5 mM Asc, but the penetration rates were higher in those without than with sperm preincubation. The addition of 0.1 mM Asc than 0.5 mM in medium with Fe$^{2＋}$ were significantly (P＜0.05) higher on acrosome reaction at 2 h after sperm preincubation. No significant differences, however, were observed in penetration rates among the concentrations of Asc. On the other hand, when preincubation medium containing the Asc was supplemented with 0.1mM Fe$^{2＋}$, the percentage of spermatozoa acrosome-reacted were significantly (P＜0.05) higher than in medium wothout Fe$^{2＋}$, on the contrary, the penetration rate was significantly (P＜0.05) low during in-vitro fertilization. These findings indicate some apparent effects of Fe$^{2＋}$ or Asc addition on acrosome reaction and the fertilizing potential by sperm preincubation.

• 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.