• Journal of Embryo Transfer
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• v.24 no.3
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• pp.199-205
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• 2009

This study was carried out to investigate the effective genetic resources preservation system using the frozen boar semen. The porcine oocytes were matured for 44 hours in NCSU-23 medium with or without 10% Porcine Follicle Fluid (PFF), 0.5 ${\mu}g/ml$ porcine FSH, 0.5 ${\mu}g/ml$ equine LH, 1.0 ${\mu}g/ml$ 17 $\beta$-estradiol ($E_2$) and 10 ng/ml Epidermal Growth Factor (EGF) under mineral oil at $38.5^{\circ}C$ in humidified atmosphere of 5% $CO_2$ in air. After 44 h of culture, the oocytes were inseminated with frozen-thawed semen and fresh semen prepared with mTBM medium for 6 h. Later, set of 50 presumptive zygotes were transferred into 4-well dish (500 ${\mu}l$) of IVC medium. for embryos freezing, slow-freezing and vitrification methods were used as a cryopreservation. Differences among treatments were analyzed using General Linear Model Procedure by SAS Package (version 6.12) differences were considered significant when p<0.05. Following IVF and IVC, the rates of cleavage and blastocysts formation were significantly higher (p<0.05) in hormone supplemented group than that of hormone-free group (25.7 vs, 12.1). The development rates to cleavage and blastocysts were significantly higher in PZM-5 group than NCSU-23 group (60.3%, 46.6% vs 27.4%, 11.1%). Further improvement was achieved when PZM-5 was supplemented with FBS. Cleavage rates was significantly higher in fresh semen source group than frozen semen (66.7% vs 43.7%). However in blastocysts rates was similar two groups. Post-thaw survival rates of embryos were 1.2% and 2.2% in slow-frezing and vitrification groups, respectively. The results of our study suggest that it is still possible to improve the culture conditions and boar semen cryopreservation for enhance reproductive technology and animal genetic resources conservation.

• Asian-Australasian Journal of Animal Sciences
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• v.22 no.4
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• pp.507-515
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• 2009

A series of experiments were conducted to determine the suitable freezing and thawing temperatures for the freezing of boar semen in 5 ml maxi-straws. The ultrastructure, in vitro fertilization (IVF) and artificial insemination (AI) of frozen-thawed semen were also be evaluated. The 5 cm freezing height gave the best results not only in post-thaw motility rate (54.00%), but also in normal acrosome morphology rate (NAR) (80.23%). There was no significant difference in the post-thaw motility between different thawing temperatures and corresponding thawing times (p>0.05); the group of $52^{\circ}C$ and 25 s gave the highest motility rate (45.00%). As a whole, not only from the motility but also the NAR, thawing at $42^{\circ}C$ was better than the other two treatments. In the freezing packages, 5 ml maxi-straw gave a little lower mobility (40%), viability rate (49.58%), plasma membrane integrity rate (53.91%) and NAR (52.65%) than the 0.25 ml straw, but there was no significant difference between the two straw volumes (p>0.05). The IVF capacity of frozen-thawed semen in this experiment was similar to fresh semen. From ultrastructure observation, the main damage to boar spermatozoa after freezing was seen in the acrosome, such as swelling and formation of vesicles. After AI in recipient Shanghai White sows, frozen-thawed semen from 5 ml maxi-straws and pellets produced 72.2% and 80% conception rate and 7.8 and 8 litter sizes, respectively, and there was no significant difference between the 5 ml maxi-straw and the pellet (p>0.05).

• Journal of Embryo Transfer
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• v.24 no.4
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• pp.275-279
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• 2009

The beneficial effect of glycerol as a cryoprotectant, especially for sperm cryopreservation, has been shown in many studies. However, glycerol is toxic to living cells, and boar sperm in particular show greater sensitivity to glycerol than sperm from other domestic animals. Amides have been studied as alternative cryoprotectants for freezing stallion sperm. Sperm frozen in methylformamide or dimethylformamide as cryoprotectants show similar motility when thawed compared with sperm frozen in glycerol. We evaluated the cryoprotective effects of dimethylformamide on boar sperm freezing. To test the effect of amides, the concentration of boar semen was adjusted to $10^9sperm/mL$, and seminal plasma was removed using Hulsen solution. After centrifugation, the pellet was diluted in modified-Modena B extender. Lactose-egg yolk (LEY) extender was used as the cooling extender. The freezing extender was madeed aaddition of the optimal amount of glycerol and amides to LEY-Glycerol-Orvus ES Paste extender, and this extender was used for the second dilution. Diluted sperm were frozen in liquid nitrogen using the 0.5 mL straw method. Sperm frozen in extender with glycerol as a cderol were compared with those frozen in extender including the different amides. Sperm were tested for motility, viability, the sperm chromatin structure assay, and normal apical ridge after thawing. The percent of motile sperm diluted in glycerol was as high as that in the stallion study (61%). Dimethylformamide showed positive effects on sperm quality and was better than glycerol. Methylformamide provided similar sperm quality as glycerol. Therefore, dimethylformamide is useful for reducing cryoinjury in boar sperm and is expected to be useful as an alternative cryoprotectant.

• Journal of Embryo Transfer
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• v.11 no.1
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• pp.81-83
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• 1996

This study was carried out to investigate the effect of sperm concentration of 5ml maxi-straw on farrowing rate and number of pigs born alive per litter in deep frozen boar semen. We did not find out the effect of sperm concentration on post-thaw sperm motility and NAR acrosome. However, farrowing rate and number of pigs born alive per litter of 7. 5 x 10˚ /5ml and 10.0 x 10˚ /5m1 sperm concentrations were higher than those of 5. 0 /10˚ /5ml sperm concentration.

• Asian-Australasian Journal of Animal Sciences
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• v.15 no.11
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• pp.1553-1558
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• 2002

This study was carried out to obtain informations regarding the effect of N-acetyl-D-glucosamine in the LEY (lactoseegg yolk) diluent according to incubation time in 5 ml maxi-straw and the effects of freezing rate, thawing temperature and thawing time in the LEN (lactose-egg yolk and N-acetyl-D-glucosamine) diluent on acrosome morphology and motility of frozen-thawed boar sperm. The study showed that the LEN diluent was higher post-thaw NAR (normal apical ridge) acrosome than the LEY diluent for 0.5 h incubation at 37$^{\circ}C$. However, there were no differences between the LEN and LEY diluents on post-thaw sperm motility according to incubation time. The straws frozen from 5.0 cm (20$^{\circ}C$/min) to 17.0 cm (1$^{\circ}C$/min) above the liquid nitrogen surface did not show any significant differences on post-thaw sperm motility. However, the straws frozen above 5.0 cm from the liquid nitrogen surface were higher NAR acrosome than those frozen above 17.0 cm. The post-thaw percentages of motile sperm and NAR acrosome were significantly higher (p<0.05) for the maxi-straws submerged for 40 or 45 sec in a 52$^{\circ}C$ water bath than for 30, 35, 50 or 55 sec. The mean sample temperatures of maxi-straws after 40 or 45 sec submersion were 20.7 or 26.4$^{\circ}C$. In conclusion, the sample temperature of the thawed semen was very important for post-thaw sperm survival in the LEN diluent of 5 ml maxi-straw. When the temperature of the thawed semen was 20.7$^{\circ}C$, the percentages of motile sperm and NAR acrosome were highest.

• Korean Journal of Animal Reproduction
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• v.23 no.2
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• pp.165-174
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• 1999

Boar semen can be frozen successfully. However, there is a large variability in the extent of damage boar semen samples experiences during cryopreservation. This experiment was undertaken to find out factors that affect a post-thaw viability of boar spermatozoa. For this purpose, cryodiluents(BF5, LEY, Soejima and M-Soejima), cryoprotectants(glycerol. ethylene glycol, and propylene glycol), pre-freezing method(dryice-pellet, dryice-straw and L$N_2$vapour-st-raw) and total time required for freezing(2. 5, and 7 h) were compared as a factors. To investigate quality of semen during freezing process, motility(%), normal apical ridges(%, NAR), and proportion of living sperm(%) by flow cytometic analysis were assessed after collection, cooled, pre-frozen and post-thawing. Post-thaw motility of semen diluted with M-Soejima was 52.0%, respectively. When heparin, caffeine or heparin＋caffeine was added to 2nd cryodiluent of M-Soejima during freezing process, the highest motility after thawing was shown at the addition of caffeine (2mM), with 61.7$\pm$2.9% of motility. M-Soejima with heparin or caffeine was significantly higher than that of controI(p＜0.05). The result using glycerol(Gly), ethylene glycol(EG), propylene glycol(PG), and their mixture (Gly＋EG and Gly＋PG) as cryoprotectants, the highest motility was shown at the mixture treatment with Gly plus PG. However, the highest proportion of live spermatozoa was shown at Gly＋EG, there was no significantly difference among treatments(p＞0.05). When semen was pre-frozen with three manners(dryice-pellet, dryice-straw, and L$N_2$ vapor-straw), motility(%) of post-thaw spermatozoa was the highest in the L$N_2$ vapor-straw pre-freezing method of M-Soejima cryodiluent with 57.5% of motility, For a simple, economical and timesaving approach to freezing boar semen, total time required for freezing were 2, 5, and 7 hours, post-thaw motility were 43.8, 45.0 and 38.8%, NAR were 19.5, 22.7 and 28.5%, and viability were 20.8, 19.9 and 22.1%, respectively. This data suggests that boar semen diluted with M-Soejima cryodiluent contained caffeine, using mixture of glycerol and propylene glycol or ethylene glycol as cryoprotectants, frozen with 2 hours, can be taken better motility, NAR, and proportion of live spermatozoa.

• Asian-Australasian Journal of Animal Sciences
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• v.15 no.4
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• pp.500-503
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• 2002

This study was carried out to investigate the effects of season influencing semen characteristics, frozen-thawed sperm viability and testosterone concentration in Duroc boars. There were no significant differences in the semen volume and sperm concentration of Duroc boars among spring, summer, autumn and winter. However, the pH of sperm-rich and sperm-poor fractions in autumn and winter season was higher than in spring and summer season in Duroc boars. Sperm motility and normal acrosome of raw semen in Duroc boars did not differ significantly among spring, summer, autumn and winter. However, motility and normal acrosome of frozen-thawed sperm were higher in spring season than in summer, autumn and winter. Serum testosterone concentrations in Duroc were higher in spring than summer, autumn and winter. In conclusion, when serum testosterone concentrations were higher in seasons, frozen-thawed sperm viability in Duroc boars were higher.

• Reproductive and Developmental Biology
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• v.40 no.4
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• pp.33-37
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• 2016

This study was conducted to evaluate effect of ${\alpha}$-linolenic acid (ALA) and bovine serum albumin (BSA) on viability, acrosome reaction and mitochondrial intact in frozen-thawed boar sperm. The boar semen was collected by gloved-hand method and cryopreserved using freezing extender containing 3 ng/mL ALA and/or $20\;{\mu}g/mL$ BSA. Cryo-preserved boar sperms were thawed in $37^{\circ}C$ water-bath for 45 sec to analysis. Viability, acrosome reaction, and mitochondrial intact were analyzed using flow cytometry. In results, viability of frozen-thawed boar sperm was significantly higher in only ALA+BSA supplement group than control group (p<0.05), whereas there was no difference either in ALA or BSA supplement. However, acrosome reacted sperm in both of live and all sperm population were significantly decreased in all treatment groups than control (p<0.05). Interestingly, mitochondrial intact of boar sperm was enhanced in ALA and ALA+BSA groups compared with control (p<0.05). In this study, we showed that supplementation of ALA and BSA in freezing extender enhanced the sperm viability, mitochondrial intact and decrease acrosomal membrane damage. In conclusion, our findings suggest that quality of frozen-thawed sperm in mammalians could improve by using of ALA and BSA.

• Proceedings of the KSAR Conference
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• 2001.03a
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• pp.86-86
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• 2001

The aims of this work were to determine the acrosin activity and to evaluate the structural and functional integrity of AS boar spermatozoa. The acrosin activity of spermatozoa were 5.40, 4.10 and 3.40 mIU/10$^{6}$ sperm in raw, extended and frozen semen respectively , which differed significantly each other (P<0.05). After the raw and extended semen were exposured to cold and thermal shock, the acrosin activities of spermatozoa in the raw semen were 5.39, 5.21 and 5.29 mIU/10$^{6}$ sperm for control (non-shock), cold shock and thermal shock, and those of extended semen were 4.21, 3.98 and 4.00 mIU/10$^{6}$ sperm. This value among treatments did not differ significantly. The acrosin activities of spermatozoa in the extended and stored semen were 3.27, 3.52, 3.46 and 3.23 mIU/10/suup 6/ sperm, while hypo-osmotic test(HOST) values were 56.5%, 64.7%, 66.0% and 56.0%, following 4 days storage at 4$^{\circ}C$, 17$^{\circ}C$ , $25^{\circ}C$ and 37$^{\circ}C$, respectively. The results at 17$^{\circ}C$ and $25^{\circ}C$ appeared to be best compared with the other storage temperatures.

• Reproductive and Developmental Biology
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• v.29 no.3
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• pp.149-154
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• 2005

The purpose of this study was the analysis of sperm ability in Specific Pathogen Free (SPE) miniature pig for production of bio-organ. The collected semen was diluted with extender and stored at $17^{\circ}C$t for up to 7 days. The semen samples were evaluated at 0, 1, 3, 5, and 7 days of storage for analysis of sperm ability. Sperm ability was evaluated by examining viability, progressive motility, sperm abnormality and intensity of the sperm membrane. Also, the semen was processed according to the convenient freezing method, and frozen-thawed sperm was evaluated by examining viability, capacitation and acrosome reaction using chlortetracycline (CTC) staining. Motility of spermatozoa of SPF miniature pig was significantly (P<0.05) lower on 3 days or later compared to the Duroc, Yorkshire and Landrace in domestic boar. The percentage of abnormal spermatozoa of Landrace were significantly (P<0.05) higher than in SPF miniature pig, Duroc and Yorkshire that had a similar percentage on 5 or 7 days of sperm storage. The percentage of spermatozoa with coiled tail decreased during the storage period but there were no significant difference. On the other hand, viability of frozen-thawed spermatozoa had a significantly (P<0.05) lower in SPF miniature pig than in other domestic boars. CTC patterns had no significant difference, but SPF miniature pig had higher percentage of capacitated spermatozoa and lower percentage of acrosome-reacted it than domestic boars. Therefore, this study suggest that it is necessary to develop the suitable extender and freezing methods methods for the high viable rate and fertilizing ability in vitro.