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

• Journal of Animal Reproduction and Biotechnology
• /
• v.37 no.2
• /
• pp.113-120
• /
• 2022

Sperm cryopreservation is a fundamental process for the long-term conservation of livestock genetic resources. Yet, the packaging method has been shown, among other factors, to affect the frozen-thawed (FT) sperm quality. This study aimed to develop a new mini-straw for sperm cryopreservation. In addition, the kinematic patterns, viability, acrosome integrity, and mitochondrial membrane potential (MMP) of boar spermatozoa frozen in the developed 0.25 mL straw, 0.25 mL (minitube, Germany), or 0.5 mL (IMV technologies, France) straws were assessed. Post-thaw kinematic parameters were not different (experiment 1: total motility (33.89%, 32.42%), progressive motility (19.13%, 19.09%), curvilinear velocity (42.32, 42.86), and average path velocity (33.40, 33.62) for minitube and the developed straws, respectively. Further, the viability (38.56%, 34.03%), acrosome integrity (53.38%, 48.88%), MMP (42.32%, 36.71%) of spermatozoa frozen using both straw were not differ statistically (p > 0.05). In experiment two, the quality parameters for semen frozen in the developed straw were compared with the 0.5 mL IMV straw. The total motility (41.26%, 39.1%), progressive motility (24.62%, 23.25%), curvilinear velocity (46.44, 48.25), and average path velocity (37.98, 39.12), respectively, for IMV and the developed straw, did not differ statistically. Additionally, there was no significant difference in the viability (39.60%, 33.17%), acrosome integrity (46.23%, 43.23%), and MMP (39.66, 32.51) for IMV and the developed straw, respectively. These results validate the safety and efficiency of the developed straw and highlight its great potential for clinical application. Moreover, both 0.25 mL and 0.5 mL straws fit the present protocol for cryopreservation of boar spermatozoa.

• Journal of Embryo Transfer
• /
• v.29 no.4
• /
• pp.397-401
• /
• 2014

The boar sperm has more lipid droplets and specialty of seminal plasma compared with other species, causing difficulties of freezing sperm and decreases for the utilization of frozen semen into the artificial insemination. However, several studies reported significant results for the recovery of sperm motility and reproductive by addition of cryoprotectants and seminal plasma after thawing. This study was designed to investigate the effects of supplementation of trehalose or glycerol in the LEY (lactose and egg yolk in BTS) solution for the conventional freezing and vitrification process. Two boars aged 16 months were used to collect semen for 2 times in a week. The samples were allotted to 3 freezing solutions (LEY + glycerol 10.5% + OEP 1.5%, LEY + trehalose 1M + OEP 1.5%, and sucrose 1.5M + trehalose 1 M + OEP 1.5%) after centrifugation at 800 g for 10 minutes. Semen was equilibrated in freezing solutions for 10 minutes and injected into plastic straws with 2~3 air bubbles to minimize freezing damages. Vitrification was performed to locate sperm in 5 cm above $LN_2$ for 5 minutes, and the conventional freezing was conducted with an automatic freezer. Motility and survival rates were measured by CASA (Computer assisted sperm an alyzing system) and FITC (Fluorescein isothiocyanate), respectively after thawing semen at $50^{\circ}C$ for 12 seconds. The results were analyzed by ANOVA with STATVIEW statistical program. The vitrificatioin solution (LEY + 10.5% glycerol + 1.5% OEP) presented higher motility (20.9%) than other solutions while the solution (LEY + 1M trehalose + 1.5% OEP) showed the lowest (motility : 5.2%). However, survival rates of vitrified sperms detected by FITC showed 1~4% live sperms in almost of dead sperms at all vitrification solutions' groups, but survival rate of freezing solution of LEY + 1M trehalose + 1.5% OEP LEY and LEY + 10.5% glycerol + 1.5% OEP were showed 49%, and 79%, respectively. There were differences (P<0.05) survival rate of conventional freezing in LEY + 10.5% glycerol + 1.5% OEP and LEY + 1M trehalose + 1.5% OEP and the remaining showed no differences. The results suggested that vitrified boar semen was not enough to be utilized for the artificial insemination, but it showed possibility to utilize for ICSI and conventional freezing with glycerol would be useful method for artificial insemination in pig while we choose the outstanding semen against tolerance to freezing damages.

• Journal of Animal Science and Technology
• /
• v.52 no.5
• /
• pp.407-412
• /
• 2010

The objective of this study was to determine the effects of dietary supplementation of whole garlic powder (WGP) on semen characteristics and blood antioxidant level in boars. For this study, nine Duroc boars of 12 months age were used. Semen and blood samples were collected for 13 weeks, once in each week. The boars were fed the basal diet (BD; control) or BD supplemented with 3% WGP. There were no significant differences in the semen volume and sperm concentration between control and WGP group on all collection weeks. However, total sperm number per ejaculate was higher in the WGP group than that in the control group on collection weeks 6, 7 and 8 (P<0.05). Also, on collection weeks 5, 6, 7 and 8, mean of total ejaculated sperm numbers per boar were significantly higher in the WGP group compared to control group (P<0.05). On the other hand, ejaculation frequency per boar (boar's libido) and total ejaculated sperm number per boar were significantly increased in the WGP group compared to the control group, respectively (P<0.05). Although there was no difference in polyphenol level in seminal plasma between two treatment groups, polyphenol level in blood serum was significantly higher in the WGP group on collection weeks 9, 12 and 13 (P<0.05). These results indicate that dietary supplementation of 3% WGP improves boar libido and semen productivity such as ejaculation frequency per boar, total sperm number per ejaculate, mean of total ejaculated sperm number per head, and elevate the blood level of antioxidant (polyphenol) in boar serum.

• Korean Journal of Animal Reproduction
• /
• v.20 no.2
• /
• pp.101-109
• /
• 1996

This study was done to find out the methods of long-term use of liquid boar semen in 100 ml plastic bottle for artificial insemination and to investigate differences between Lactose-Egg yolk and Biitschwiler diluents according to storage temperature, and effect of final glycerol concent ration in Lactose-Egg yolk diluent. Liquid boar semen diluted with Lactose-Egg yolk diluent showed significantly higher sperm motility (p<0.05) after 0.5 and 2h incubation at 37$^{\circ}C$,than Butschwiler diluent at all storage length when it was preserved in the 5$^{\circ}C$ refrigerator. The NAR acrosome in Lactose-Egg yolk diluent a after 0.5 and 2h incubation at 37$^{\circ}C$, respectively, during preservation periods was similar to that in Biitschwiler diluent. When liquid boar semen was preserved at 15$^{\circ}C$, liquid boar semen in the Butschwiler diluent showed significantly higher percentages of sperm motility and NAR acrosome from third day to seventh than that in Lactose-Egg yolk diluent. In the effect of final glycerol concentration of liquid boar semen in the Lactose-Egg yolk diluent, the final glycerol concentration of 2% showed higer percentages of sperm motility and NAR acrosome than that of 0, 1, 3, and 5%. Farrowing rate, litter size and average pig weight at birth did not differ significantly between Lactose-Egg yolk and But schwiler diluents. As a result of this study, we found out that liquid boar semen can be stored for 6-7 days at 5$^{\circ}C$ in Lactose-Egg yolk diluent and at 15$^{\circ}C$ in Butschwiler diluent.

• Korean Journal of Agricultural Science
• /
• v.28 no.2
• /
• pp.85-91
• /
• 2001

This study was carried out to investigate the effects of liquid boar semen on reproductive performance in swine artificial insemination. Many factors, which were breeds, time of insemination, breeding season, sperm per dose etc, have been tried to improve reproductive efficiency. Boars were raised at Swine Artificial Insemination Center in National Livestock Research Institute, Sunghwan, Chungnam, Korea. This experiment was carried out from 1995 to 2000. There were no differences in the fertility results compared with 3 breeds (Landrace, Yorkshire and Duroc), frequencies of artificial insemination (double and triple) per estrus cycle and different seasons by using liquid boar semen. There were no significant differences in conception rate, farrowing rate and litter size using 4 trials of 3.0, 2.5, 2.0 and $1.5{\times}10^9/80ml$ in liquid boar semen with 70% of motile sperm cells. We confirmed that the sperm number per dose of $1.5{\times}10^9/80ml$ could be used for commercial artificial insemination.

• Korean Journal of Animal Reproduction
• /
• v.26 no.2
• /
• pp.119-124
• /
• 2002

This study was carried out to investigate the effects of packing materials of frozen boar semen to improve reproductive performance efficiency in pig. Boars were raised at Swine Artificial Insemination Center in National Livestock Research Institute, Sunghwan, Chungnam, Korea. We compared packing protocols for frozen boar semen among 5$m\ell$ maxi-straw, 5$m\ell$ cryogenic-vial, and aluminum-pack. Cryogenic-vial packing material showed similar sperm characteristics compared with maxi-straw packing material when the sperm was frozen above 15cm from liquid nitrogen and thawed at 52$^{\circ}C$ for 190 seconds. We investigated different thawing times to find out the optimal condition of freezing and thawing protocol with cryogenic-vial. Freezing above 15cm from liquid nitrogen and thawing at 52$^{\circ}C$ for 190 seconds were the optimal protocol compared with 120 and 150 seconds. However, normal acrosome rates did not show any differences among thawing times. Post-thawing results of maxi-straw in water at 52$^{\circ}C$ for 45 seconds had better total motility and curve linear velocity than those of cryogenic-vial in water 52$^{\circ}C$ for 190 seconds. However, there were no differences on straightness and normal apical ridge of sperm between maxi-straw and cryogenic vial. Non-return rate, farrowing rate and litter size of sows inseminated with frozen boar semen of commercial farms were higher in the maxi-straw than cryogenic-vial, but there were no significant differences between maxi-straw and cryogenic-vial. In conclusion, there were no significant differences between maxi-straw and cryogenic-vial and so, we may replace cryogenic-vial packing method instead of maxi-straw packing method by improvement of freezing and thawing rate.

• Korean Journal of Animal Reproduction
• /
• v.25 no.4
• /
• pp.327-337
• /
• 2001

This study investigated the effects of superoxide dismutase (SOD) on lipid peroxidation and fertilizing ability in vitro of boar spermatozoa frozen-thawed. The percentages of motile sperm were highest when SOD of 10 units/$m\ell$ was added to washing medium for spermatozoa. However, the rates of motile sperm were not significantly different in different concentrations of SOD. On the other hand, the motile rates of sperm washed with SOD were lower in sperm inculbated for 120 min than 30 min regardless of the different concentrations of SOD. The percentage of spermatozoa that reached acrosome reaction were increased with incubation periods prolonged. No significant differences, however, were observed in acrosome reaction rates between sperm incubated with and without SOD supplementation for 0, 60 and 120 min. When oocyies inseminated with different concentrations of SOD, the penetration rates were significantly (P＜0.05) higher in medium with 1 unit/$m\ell$ than 0, 10 and 100 units/$m\ell$ of SOD. However, the proportions of polyspermit oocytes were significantly (P＜0.05) lower in medium with 10 and 100 units/$m\ell$ than 0 unit/$m\ell$ of SOD. In another experiment, the sperm suspension were also treated with different concentrations of SOD and were assayed far sulfhydryl(-SH) group content. In the groups treated with 100 units/$m\ell$ of SOD, sperm-SH group were higher than another groups. However, sperm-SH group content were not siginificantly different in spermatozoa treated with different concentrations of SOD. Under the same conditions, the lipid peroxidation of sperm was evaluated on the basis of malondialdehyde production. The addition of SOD to sperm suspension decreased the formation or malondialdehyde. However, there were not significantly different in sperm treated with different concentrations of SOD. The activity of sperm binding to zona pellucida was also evaluated through binding to salt-stored porcine oocytes. The sperm binding to zona pellucida were gradually increased with SOD concentrations added. The number of spermatozoa binded to zona pellucida were significantly (P＜0.05) higher in medium with 100 units/$m\ell$ than 0 units/$m\ell$ of SOD. These findings suggested that SOD cause an enhancement penetrarion ability and sperm zona binding in boar spermatozoa frozen-thawed.

• Journal of Embryo Transfer
• /
• v.20 no.1
• /
• pp.43-48
• /
• 2005

This experiment was carried out to investigate the extender, cooling rate and concentration of glycerol for freezing of boar semen. The result obtained were summarized as follows: 1. Optimal cooling rate was $0.17\~022^{\circ}C/min$ from 25 to $5^{\circ}C$ in LEY extender on the viability and normal acrosome after thawed. 2. The LEY extender was effective in protecting frozen boar semen from cold shock among the extenders(p<0.001, respectively). 3. The sperm viability and normal acrosome rates after thawing was showed greater in the 3 or $4\%$ of glycerol concentration than $2\%$ in LEY extender. 4. Viability of sperm was higher when both 15mM of fructose and 3 or $4\%$ glycerol were added to the LEY extender compared with other concentrations of fructose and glycerol were added it(p<0.001).

• Reproductive and Developmental Biology
• /
• v.35 no.3
• /
• pp.203-207
• /
• 2011

The objective of this study was to determine the effect of semen extenders on the motility, viability and fertility in vitro of spermatozoa during storage of fresh boar semen diluted in different commercial extenders used for pig artificial insemination (AI). In this experiment, semen were diluted in Androhep plus, Beltsville Thawing Solution (BTS), Modena, Seminark and Vitasem LD. Five ejaculates were collected from three Duroc boars and sub-samples were diluted ($30{\times}10^6$ spermatozoa/ml) in different extenders. Semen was stored at $170^{\circ}C$ for 10 days. Sperm motility and viability was assessed using Computer-Assisted Semen Analysis (CASA) and flow-cytometry on 1, 3, 5 and 10 day post collection The motility of spermatozoa stored in different extenders was gradually decreased by increasing the duration of storage of semen. However, there was not significant1y different in the sperm motility and viability among other extenders. On the other hand, the in vitro-matured oocytes were fertilized and cultured in vitro to assess the fertility of boar spermatozoa stored for 3 and 10 days in different extenders. The percentage of morula and blastocyst were taken as indicators of fertility in vitro of spermatozoa. Therefore, there were no differences in the rate of embryos developed to the molular and blastocyst stage. There were no differences in the motility and fertility in vitro among 5 kinds of commercial boar semen extenders.