• Asian-Australasian Journal of Animal Sciences
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• v.31 no.6
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• pp.842-850
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• 2018

Objective: Several studies have reported the development of new molecular methods for the prognosis and diagnosis of male fertility based on biomarkers aimed at overcoming the limitations of conventional male fertility analysis tools. However, further studies are needed for the field application of these methods. Therefore, alternative methods based on existing semen analysis methods are required to improve production efficiency in the animal industry. Methods: we examined the possibility of improving litter size in various pig breeds using combined Hoechst 33258/chlortetracycline fluorescence (H33258/CTC) staining. The correlation between field fertility and capacitation status by combined H33258/CTC staining in different ejaculates spermatozoa (n = 3) from an individual boar (20 Landrace, 20 Yorkshire, and 20 Duroc) was evaluated as well as overall accuracy. Results: The acrosome reacted (AR) pattern after capacitation (%) was positively correlated with the litter size of Landrace, Yorkshire, and Duroc pigs and the overall accuracy was 75%, 75%, and 70% in Landrace, Yorkshire, and Duroc pigs, respectively. The difference (${\Delta}$) in AR pattern before and after capacitation was positively correlated with the litter size of Landrace, Yorkshire, and Duroc pigs and the overall accuracy was 80%, 65%, and 55% in Landrace, Yorkshire, and Duroc pigs, respectively. However, the difference (${\Delta}$) in capacitated (B) pattern before and after capacitation was negatively correlated with the litter size of Landrace pigs and the overall accuracy was 75%. Moreover, average litter size was significantly altered according to different combined H33258/CTC staining parameters. Conclusion: These results show that combined H33258/CTC staining may be used to predict male fertility in various breeds. However, the selection of specific efficiency combined H33258/CTC staining parameters requires further consideration. Taken together, these findings suggest that combined H33258/CTC staining may constitute an alternative method for predicting male fertility until such time as fertility-related biomarkers are further validated.

• Clinical and Experimental Reproductive Medicine
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• v.36 no.1
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• pp.45-53
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• 2009

Objectives: To determinate the optimal culture condition to maintain lifespan in human sperm, we evaluated the effect of different temperature on sperm motility and viability up to 5 days in normal specimens. Methods: Ejaculated semen samples with normal semen parameters were gently washed in HEPES buffered Tyrod's-Albumin-Lactate-Pyruvate (HTALP) media. Each 5 ml of HTALP + 0.3% albumin with $1{\times}10^6$ sperm/ml was incubated for 5 days in $37^{\circ}C$, $22^{\circ}C$, and $4^{\circ}C$. The sperm motility and kinematics were analyzed using computer assisted sperm analysis (CASA), membrane integrity was assessed by hypoosmotic swelling test (HOST), and capacitation status was evaluated by chlorotetracycline (CTC) fluorescence pattern. Each parameter was measured on day 1, 3, and 5, respectively. Results: The motility, viability and live/uncapacitated pattern were demonstrated significantly in temperature- and time-dependent difference (p<0.05). While the sperm cultured for 1 day in each temperature was not significantly different, the sperm cell kept in $22^{\circ}C$ after 3 days were preserved sperm motility, viability, membrane integrity, and F pattern better than in other culture temperatures. Conclusions: HTALP can be used a basic medium for culture and longevity preservation, and sperm cell kept at $22^{\circ}C$ is beneficial for assisted reproductive techniques.

• Reproductive and Developmental Biology
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• v.36 no.3
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• pp.163-166
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• 2012

This study was conducted to determine the relationship between elapsed time after semen preservation on the changes of bacteria and semen quality. Semen was diluted with BTS(Beltsville Thawing Solution) extender without antibiotic for 7 days and sperm parameter and fertility were measured. Sperm motility was measured by CASA and total bacteria number was counted after 22~24 hr incubation from counting agar plate in which sperm dilute to $10{\sim}10^6$ in 0.9% saline solution and inoculate to agar. Acrosomal integrity was measured by Chlortetracycline (CTC) staining. CTC patterns were uniform fluorescence over the whole head (pattern F), characteristic of incapacitated acrosome-intact spermatozoa; fluorescence-free band in the post-acrosomal region (pattern B), characteristic of capacitated acrosome-intact spermatozoa; and almost no fluorescence over the whole head except for a thin band in the equatorial segment (pattern AR), characteristic of acrosome reacted spermatozoa. Total number of bacteria was significantly increased (p<0.0001) 3 days after preservation. Sperm motility, viability, and morphological abnormality on elapsed time after preservation were lower from 5 ($77.24{\pm}6.47$, p<0.001) and 7 days ($77.24{\pm}6.47$, p<0.001) after preservation compared to 1 ($15.71{\pm}7.18$) and 3 days($18.39{\pm}7.22$) after preservation, respectively. Sperm viability was significantly lower ($53.25{\pm}35.03$, p<0.0001) at 7 days after preservation. Morphological abnormality of sperm was lower (p<0.001) at 1 ($15.71{\pm}7.18$) and 3 ($18.39{\pm}7.22$) days compared to 5 ($21.84{\pm}7.91$) and 7 ($22.59{\pm}9.93$) days after preservation. Acrosomal integrity and capacitation rate (pattern F) were significantly lower (p<0.001) from 5 days after preservation. Based on the data we obtained from this study suggested that semen preserved more than 5 days without antibiotic would not recommend use for artificial insemination.

• Journal of Embryo Transfer
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• v.26 no.3
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• pp.187-193
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• 2011

This study was carried out to establish most suitable freezing condition, to evaluate the different glycerol concentration of freezing and thawing rates on motility, viability, membrane integrity and acrosome intecrity of frozen Korean Jeju Black Bull spermatozoa, Semen was collected from a Korean Jeju Black Bull using an artificial vagina and transported to the laboratory. The semen was extended gradually 1:5 then cooled slowly for 2 hrs to 4$^{\circ}C$. The semen was diluted 1:1 with cryoprotectant extenders (3%, 5% and 7% glycerol) and equilibrated for 2 hrs at cold chamber and packed to 0.5 ml straws. The semen straws were located above 3 cm of liquid nitrogen for 5 minutes, above 5 cm for 10 min and above 8 cm for 10 min. And then the frozen straw was plunged into LN$_2$. The presented straws were examined the viability and motility after thawed at 37$^{\circ}C$ water bath. The viability and membrane integrity immediately post-thawing were significantly higher in samples frozen in 7% glycerol than 3% and 5% glycerol (p<0.05). After CTC staining to assess acrosome integrity, F pattern was significantly increased, but B pattern was significantly decreased in 7% glycerol (p<0.05). Freezing distance of 5 cm from liquid nitrogen and pre-cooling for 10 min yield better survival and membrane integrity, but not significant difference. However, AR pattern according to CTC staining was significantly decreased in 3 cm for 5 min.

• Proceedings of the Korean Society for Technology of Plasticity Conference
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• 1999.08a
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• pp.57-65
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• 1999

This document illustrates the way to control of coiling temperature(CTC) of stip tail part in minimill process. The coiling temperature (CT) is very important fact in hot rolling process because the mechnical properties of strip depend on it. In mini-mill pocess, the speed pattern of rolling is different from that of conventional hot rolling. We have a lot of difficulties in controlling the coiling temperature at strip tail part, because after the strip tail is passed out the final stand, it's impossible to control the coiling temperature by using coiling speed. So we have CT deveiation in this gauge, about 160$^{\circ}C$ below in comparison with target CT. It's clear that deviation of mechanical properties(tensile strength, vield ratio etc) of strip to direction of length is so large, so we could not produce middle carbon steel, like this temperature condition. New coiling temperature control of strip especially in strip tail was developed. An innovative method for calculate the deceleration speed of strip tail has been implemented in CTC program, so called

• Asian-Australasian Journal of Animal Sciences
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• v.20 no.3
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• pp.351-358
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• 2007

Carbohydrate-protein interactions are known to be important in gamete interactions. Several evidence indicated that a fucose-containing sulfated polysaccharide fucoidan was potential inhibitor of fertilization in vitro and thus fucose seemed to be part of the recognition signal of gamete interaction in mammals. In recent investigation we found that ${\alpha}$-L-fucosidase activity was present in boar spermatozoa and it was related to sperm binding to and penetration into zona pellucida (ZP) in vitro. The objective of this study was to determine the effects of monosaccharide L-fucose and polysaccharide fucoidan on sperm ${\alpha}$-L-fucosidase activity and relation to sperm-oocyte interaction in pig. Results indicated that the activity of sperm ${\alpha}$-L-fucosidase was largely inhibited (62%) when sperm suspension was treated with monosaccharide L-fucose. It also significantly inhibited the number of sperm binding to ZP (32%) and penetration into zona-intact oocytes (72%), but did not inhibit penetration into zona-free oocytes when fertilization medium contained L-fucose. The chlorotetracycline (CTC) assessment showed that L-fucose did not affect induction of sperm capacitation and acrosome reaction. In contrast, the activity of sperm ${\alpha}$-L-fucosidase was not inhibited when sperm suspension was treated with polysaccharide fucoidan but sperm-ZP binding was greatly inhibited (85%) and completely blocked sperm penetration into zona-intact or zona-free oocytes. The CTC assessment showed that fucoidan increased the F pattern and decreased the AR pattern sperm. These results suggested that the different inhibitory mechanisms were present between monosaccharide L-fucose and polysaccharide fucoidan on sperm-oocyte interaction, the inhibition effect of ${\alpha}$-L-fucose on sperm binding and penetrating into ZP caused sperm ${\alpha}$-L-fucosidase inhibited by ${\alpha}$-L-fucose.

• Reproductive and Developmental Biology
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• v.28 no.4
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• pp.261-265
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• 2004

The present study was conducted to assess sperm characteristics in miniature-pig. The semen samples were transported to the laboratory at 17℃ within 3 hours after collection. The extended semen was stored at 17℃, and sperm quality was evaluated at 0, 1, 3, 5 and 7 days after storage. The semen volume of miniature-pig (62±22㎖) was significantly (p<0.05) lower than that of Duroc (155±25㎖) and Yorkshire (154±23㎖). Significant differences were also observed in sperm concentrations. During 3 days of storage, sperm viability did not differ among miniature-pig, Duroc and Yorkshire. However, the viability was significantly (p<0.05) lower in miniature-pig than in Duroc and Yorkshire semen after Day 3 of storage. In abnormality, acrosome intactness and intensity, there were no differences among miniature-pig, Duroc and Yorkshire semen. On the other hand, the viability of frozen-thawed sperm in miniature-pig was significantly (p<0.05) lower than in that of Duroc and Yorkshire. This study also examined CTC patterns in frozen-thawed spermatozoa. The rates of AR pattern were higher in miniature-pig than in Duroc and Yorkshire. However, no difference was found in F, B and AR patterns. The results of present study suggest that further research is necessary to develop of semen extender and freezing methods to improve sperm quality in miniature-pig.

• Reproductive and Developmental Biology
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• v.29 no.4
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• pp.247-252
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• 2005

The purpose of this study was to assess sperm quality during in vitro storage of miniature-pig semen in order to determine which extender should be used and how extender can be diluted for in vitro storage of miniature-pig semen. Freshly ejaculated miniature-pig's semen was diluted with same volumes of Beltsville Thawing Solution (BTS), Androhep, Modena, Mulberry III and modified-Modena extenders. Sperm quality was evaluated by examining viability, motility, abnormality, acrosome intactness, intensity and capacitation status by chlorotetracycline (CTC) staining. Sperm motility decreased with storage period prolonged and differences among BTS, Androhep, Modena and Mulberry III were apparent On Day 1, approximately 80% of the sperm were motile, but motility decreased to $40\%$ at Day 7. During the 7 days of storage, sperm survival in modified-Modena B extender was higher than another extenders. However, it was not differ significantly among other extenders. The percentage of F and B patterns were not differ significantly among the extenders. However, F pattern in modified-Modena B extender was slightly higher until 3 days of storage than that of Modena extender, modified-Modena A extender and modified-Modena C extender. The percentage of AR patterns in modified-Modena B extender was slightly lower, but did not differ significantly among other extenders. The results of present study suggest that modified-Modena B was effective as new extender for in vitro storage of miniature-pig semen.

• Reproductive and Developmental Biology
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• v.31 no.3
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• pp.175-179
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• 2007

The objective of this study was to investigate the effect of thawing temperature on the sperm viability and acrosomal morphology for semen storage of miniature pig by the 0.5ml straw method. In this present study, sperm viability (SYBR-14/PI staining), membrane integrity (Hypoosmotic Swelling Test), acrosome intactness, intensity and capacitation status (chlorotetracycline staining) in frozen miniature pig sperm were evaluated after thawing at 37, 50 and $70^{\circ}C$ for 5, 10 and 45 sec, respectively. Interestingly, the results indicated that sperm thawed at $70^{\circ}C$ for 5 sec significantly (p<0.05) increased sperm viability, but lower the percentage of AR (acrosome reacted spermatozoa) pattern compared to sperm thawed at $37^{\circ}C$ for 45 sec and $50^{\circ}C$ for 10 sec. In terms of thawing condition, high temperature for a short time using the 0.5ml straw was improved cryosurvival of miniature pig semen. Therefore, appropriate thawing method for cryopreservation of miniature pig is required for increasing post-thawing viability.

• Reproductive and Developmental Biology
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• v.35 no.3
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• pp.385-390
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• 2011

The purpose of this study was undertaken to evaluate of cryopreservation efficiency in ${\alpha}$ 1,3-galactosyltransferase knock-out(GalT KO) cloned miniature pig sperm. To compare ability of frozen-thawed sperm characteristics, three different pig strains (GalT KO) cloned miniature pig, PWG miniature pig and Duroc were used. The ejaculated semen from the three pig species was diluted with same volume extender and added to LEY solution for freezing. The diluted semen was placed in 0.5 ml straws, and freezing was initiated by exposing the straws to liquid nitrogen ($LN_2$) vapours for 10 min before placing them into $LN_2$ for cryopreservation. After thawing, the sperm ability were assessed for viability (SYBR-14/PI staining), abnormality (Rose Bengal staining), and acrosome status (intactness, intensity and capacitation) (chlorotetracycline, CTC staining). The viability of frozen-thawed GalT KO pig sperm had no significant difference as compared with Duroc and PWG miniature pig sperm. However, The CTC pattern of frozen-thawed GalT KO cloned miniature pig spermatozoa showed significantly lower rates in F pattern and AR pattern (p<0.05) and significantly higher rates in B pattern than Duroc and PWG miniature pig (p<0.05). The abnormality of GalT KO cloned miniature pig sperm was significantly lower as compared to Duroc and PWG miniature pig sperm (p<0.05). In conclusion, GalT KO cloned miniature pig semen can be cryopreserved successfully and used for artificial insemination reasonably.