• 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.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.

• 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.

• Journal of Embryo Transfer
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• v.27 no.2
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• pp.107-111
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• 2012

Miniature pig sperm cryopreservation is continually researched in biotechnology for breed conservation and reproduction. It is important to control the temperature at each stage of cryopreservation and cryoprotectant. It is also necessary to find the optimal cryoprotectant concentration and chemical elements of the extender. Recently, many studies have used various cryoprotectant materials, such as dimethyl sulphoxide (DMSO), ethylene glycol (EG), antifreeze protein (AFP), amides, and glycerol. Glycerol is a commonly used cryoprotectant. However, glycerol has critical cytotoxic properties, including osmotic pressure and it can cause irreversible damage to live cells. Therefore, We focused on membrane fluidity modifications can reduce cell damage from freezing and thawing procedures and evaluated on the positive effects of trehalose to the viability, chromatin integrity, and motility of boar sperm. Miniature pig sperm was separated from semen by washing with modified- Modena B (mMB) extender. After centrifugation, the pellet was diluted with the prepared first extender. This experiment was designed to compare the effects that sperm cryopreservation using two different extenders has on sperm chromatin. The control group used the glycerol only and it was compared with the glycerol and glycerol plus trehalose extender. Sperm viability and motility were evaluated using WST1 assays and computer-assisted semen assays (CASA). Chromatin structure was examined using acridine orange staining. For the motility descriptors, trehalose caused a significant (p<0.01) increase in total motility ($57.80{\pm}4.60%$ in glycerol vs. $75.50{\pm}6.14%$ in glycerol + trehalose) and progressive ($51.20{\pm}5.45%$ in glycerol vs. $70.74{\pm}8.06%$ in glycerol + trehalose). A significant (p<0.05) increase in VAP ($42.70{\pm}5.73{\mu}m/s$ vs. $59.65{\pm}9.47{\mu}m/s$), VSL ($23.06{\pm}3.27{\mu}m/s$ vs. $34.60{\pm}6.58{\mu}m/s$), VCL ($75.36{\pm}11.36{\mu}m/s$ vs. $99.55{\pm}12.91{\mu}m/s$), STR ($54.4{\pm}2.19%$ vs. $58.0{\pm}1.63%$), and LIN ($32.2{\pm}2.05%$ vs. $36.0{\pm}2.45%$) were also detected, respectively. The sperm DNA fragmentation index was 48.8% to glycerol only and 30.6% to glycerol plus trehalose. Trehalose added group showed higher percentages of sperm motility, stability of chromatin structure than glycerol only. In this study, we suggest that trehalose is effective in reducing freezing damage to miniature pig sperm and can reduce chromatin damage during cryopreservation.

• Journal of Embryo Transfer
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• v.21 no.3
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• pp.263-271
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• 2006

The purpose of this study was undertaken to compare ability of frozen-thawed sperm characteristics between two strains (miniature pig and Duroc). The semen was collected by gloved-hand method into a pre-warmed ($37^{\circ}C$) thermos bottle. The semen 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. The frozen-semen straw were thawed at 20, 37 and $50^{\circ}C$ for 1 min, 45 sec and 10 sec within water-bath. The semen sample were evaluated at 0, 3, 6, 9, and 12 h after incubation at $37^{\circ}C$ for analysis of sperm ability. Abnormality of spermatozoa in miniature pig was significantly (p<0.05) higher than that in Duroc at 0, 9 and 12 h of post-thawing incubation after frozen-thawing. The percentage of F-patterned spermatozoa in miniature pig was significantly (p<0.05) lower, while the percentage of AR (acrosome reacted spermatozoa) pattern was higher in the miniature than in the Duroc. On the other hand, there was no significant difference in the viability of spermatozoa thawed at different temperature ($20^{\circ}C\;and\;37^{\circ}C$) between two species, but the viability in miniature pig was higher (p<0.05) than in Duroc when sperm was thawed at $50^{\circ}C$. In conclusion, this study suggest that suitable freezing method for miniature pig semen is required for increasing post-thawing viability and fertilization capacity.

• Journal of Embryo Transfer
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• v.29 no.1
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• pp.83-90
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• 2014

The main purpose of this study is to estimate the effect of adding Tea-N-Tris (TES) to the freezing buffer for miniature pig sperm. In particular, we attempted to identify the association between the MMPs expression and the fertility and viability of frozen sperm from each extender (LEY (Lactose Egg-Yolk), TLE (TES + LEY), TFGE (TES + Fructose + Glucose Egg-Yolk)). In accordance with this, Hypoosmotic Swelling Test (HOST) respond test was the lowest among sperms frozen in LEY while the highest HOST respond was observed among sperms frozen in TLE. Furthermore, we observed MMPs expression in all sperm groups, with pro-MMP showing lower expression than active MMPs. The expression of MMP-9 and MMP-2 was the highest in sperms frozen in LEY, Meanwhile, sperms from the TFGE and TLE group showed lower level of MMP-9 and MMP-2 expression in the order of TLE being the lowest. LEY group showed lower rate of blastocyst development than the TES supplement group, although the difference was not statistically significant. Meanwhile the rate of blastocyst development appeared similar when sperms from TLE and TFGE group were used for IVF. Together, these results indicate that adding Tea-N-Tris to the sperm freezing buffer only suppresses MMPs protein activation but also maximize in-vitro fertility, providing a means to improve the success rate in the in vitro manipulation of miniature pig sperm.

• Reproductive and Developmental Biology
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• v.35 no.1
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• pp.85-91
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• 2011

The objective of this study was to evaluated the efficiency on sperm cryosurvival and ability of in vitro fertilization using Triladyl and Lactose Egg-Yolk(LEY) as extenders for cryopreservation of separated sperm by 65% percoll in miniature pig. Sperm viability was measured with SYBR-14/PI double stained sperm by flow cytometry. Ability on embryo cleavage rate and blastocyst development were observed by in vitro fertilization after frozen-thawing of sperm separated by 65% percoll. The experimental groups were designed that separated sperm by 65% percoll with Triladyl (ST) or LEY(SL) and unseparated sperm with Triladyl(UT) or LEY(UL) for cryopreservation. As a results, the viability was significantly(p<0.05) higher in ST(55.1%), SL(63.1%), UL(58.8%) than UT(38.2%) group. Sperm viability in SL(63.1%) group was significantly(p<0.05) higher than other experimental groups. On the other hand, embryo cleavage rate was significantly(p<0.05) higher in ST(79.1%), SL(83.2) than UT(74.1) and UL(75.7%) groups at 96h after in vitro fertilization. Blastocyst development was also significantly(p<0.05) higher in ST(21.5%), SL(20.9%) than UT(17.0%) and UL(18.8%) groups. In conclusion, cryopreservation of miniature boar sperm separated by 65% percoll were beneficial to viability and capacity on in vitro fertilization.

• Journal of Embryo Transfer
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• v.32 no.3
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• pp.249-255
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• 2017

In this experiment, we determined the effect of curcumin supplementation in freezing buffer for miniature pig sperm cryopreservation. Each ejaculate was diluted with modified Modena B extender and mixed with lactose-egg yolk (LEY extender, 80% v/v lactose solution [310 mM], 20% v/v egg yolk, and $100{\mu}g/mL$ kanamycin sulfate) and LEY-glycerol Orvus ES Paste (LEYGO, 89.5% v/v LEY, 5% v/v glycerol, 1.5% v/v Orvus ES Paste), 100 mM trehalose supplemented with 0, 10, 50, 100, and $500{\mu}M$ of curcumin from turmeric, respectively. Following equilibration, the 0.5 mL French straws were frozen and plunged into $LN_2$ tank for 7 days at least. Sperm parameter and oxidative byproducts were determined by the computer assisted sperm motility analysis (CASA) and fluorescence-activated cell sorting (FACS) as compared with each groups. Supplementation of curcumin had no effect on sperm motility, progressive motility and curvilinear velocity. However, average-path velocity and straight-line velocity were significantly higher in $10{\mu}M$ curcumin group ($100.9{\pm}8.8{\mu}m/s$, $61.7{\pm}2.9{\mu}m/s$, respectively) than control group ($77.8{\pm}3.9{\mu}m/s$, $46.4{\pm}3.0{\mu}m/s$, respectively) (p < 0.05). In addition, the level of the O2 radical and H2O2 were comparatively decreased in curcumin groups by evaluation of ethidium and DCF fluorescence. According to the results, curcumin can improve sperm kinetic variables and alleviate ROS induced cryoinjury to pig sperm.