• Asian-Australasian Journal of Animal Sciences
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• v.21 no.4
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• pp.494-498
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• 2008

MTT (3-(4, 5-dimethyl thiazol-2-yl)-2, 5-diphenyl tetrazolium bromide) reduction assay is a method that validates the viability of an active cell. Dehydrogenase in mitochondria converts yellow colored insoluble tetrazolium salt to purple colored water-soluble formazan. Sperm also have mitochondria in the midpiece, therefore sperm viability could be evaluated by MTT reduction assay. Several studies have already demonstrated the capability of application of the MTT reduction assay to sperm of several species in Hepes-BSA buffer. Because most liquid semen was diluted in extender like BTS (Beltsville Thawing Solution), Modena or Androhep when it is used or transferred, semen needed another dilution in Hepes-BSA buffer to assess sperm viability. In this study, we evaluated boar sperm viability especially in BTS extended semen and compared the efficiency of this test with eosin-nigrosin staining. We used the fresh BTS extended semen from a local A.I center. Semen sample was diluted to $3.0{\times}10^7$ sperms/ml in BTS. The rates of formazan production were measured in 96-well microtiter plates immediately and 1h after incubation at $17^{\circ}C$ using a spectrophotometer at wave length 560 nm. Simultaneously, split samples of the same semen were tested, using eosin-nigrosin staining to compare the efficiency of the MTT assay of sperm viability in BTS. The correlation between the results of these tests was calculated using Student-t test and ANOVA. The results revealed a strong correlation between the results of MTT reduction rate and the results that were simultaneously determined by eosin-nigrosin staining at 1 h. In conclusion, the MTT reduction test was an effective and simple method to validate sperm viability and it could be used as a simple tool to evaluate sperm viability in the local A.I center and laboratory.

• 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 Veterinary Clinics
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• v.18 no.4
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• pp.345-349
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• 2001

The aim of this study was to investigate the effect of sperm preservation according to the various kinds of commercially available semen extenders and to investigate the effect of sperm preservation according to the various temperature storages of fresh-extended porcine semen. To investigate the effect of sperm preservation according to the various kinds of commercially available semen extenders, porcine semens diluted in 3 semen extenders, Beltsville Thawing Solution(BTS), Androhep and Kiev, were cooled at $8^{\circ}C$ storage temperature with a controlled cooling rate of $2-4^{\circ}C$/h. Motility, progressive motility, normal sperm(%) and sperm morphology were assessed comparatively. In motility and progressive motility, Androhep extenders revealed better result than other extenders. In normal sperm(%) and sperm morphology, 3 semen extenders revealed similar results. To investigate the effect of sperm preservation according to the various temperature storages of fresh-extended porcine semen, porcine semens diluted in BTS extender, were cooled at 3 storage temperatures($8^{\circ}C$, $12^{\circ}C$ and $17^{\circ}C$) with a controlled cooling rate of $2-4^{\circ}C$/h. Motility, progressive motility, normal sperm(%) and sperm morphology were assessed comparatively. In motility and progressive motility, $8^{\circ}C$ treatment group revealed better result than $12^{\circ}C$ and $17^{\circ}C$ treatment groups. In normal sperm(%) and sperm morphology, 3 temperatures of treatment groups revealed similar results.

• Korean Journal of Animal Reproduction
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• v.26 no.1
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• pp.9-16
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• 2002

This study was carried out to investigate the effects of extenders such as Beltsville thawing solution(BTS), Modena and Androhep, preservation temperature and period of liquid boar semen on semen characteristics and reproductive performance. 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. The results obtained were summarized as follows. 1. Sperm motility in the samples with Androhep and BTS reduced from day 5 and in the samples with Modena reduced from day 3 of storage. pH or 3 extenders varied from 6.24 to 7.04 during day 1 to 5 of storage. Farrowing rate of sows inseminated with liquid boar semen offended with BTs, Modena and Androhep extenders did not show any differences until day f after semen collection. Sows inseminated with Androhep extender had better farrowing rates (P＜0.05) than those with Modena extender at day 1 or 5 after semen collection, but farrowing rates after AI using BTS did not differ compared to those Androhep and Modena. Litter size did not show any differences among the three extenders, but Androhep had the decreased litter size from day i of storage. 2. Motility and normal acrosome of the sperm preserved at 5$^{\circ}C$ did not show any differences until day 4 of storage, but those at 17$^{\circ}C$ changed from day 3 and 4, respectively. Farrowing rate of sows artificially inseminated with liquid boa. semen preserved at 17$^{\circ}C$ had higher, than at 5$^{\circ}C$ (p＜0.05), but there was no significant differences in litter size. Farrowing rates and litter size were decreased from day 2 and day 3 of storage at 17$^{\circ}C$, respectively. Farrowing rate of sows inseminated with the preserved semen at 5$^{\circ}C$ did not changed until day 4, but the litter size at 5$^{\circ}C$ was lower than that at 17$^{\circ}C$.

• Korean Journal of Animal Reproduction
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• v.22 no.4
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• pp.405-410
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• 1998

Boar semen extender Kp (Hankook Life-Science, Korea) was newly formulated by authors. This study was carried out to investigate the optimal concentrations of EDTA, Tris and citrate buffers in the Kp extender (Basic Kp) on the pH change during storage. And then the motility of boar sperm with the Kp pH stabilized (Modified Kp) was compared with those of commercial products imported into Korea such as BTS (Mini-tube. Germany; BTSg), BTS (Tri-bio, USA; BTSa) and Modena (SGI, USA). The pH values of all extenders were increased gradually with the storage days. Especially, the initial pH of Basic Kp was higher than that of BTSg, BTSa and Modena, and also higher than physiological pH of boar sperm (6.8∼7.5). When Basic Kp was added with various concentrations of EDTA (0, 0.63, 1.25 ＆ 2.37g/L), Tris (0, 0.18, 0.35, 0.71 ＆ 1.42g/L) and Citrate (0, 0.75, 0.81, 1.00, 1.25 ＆ 1.50g/L) buffers for pH down-regulation and stabilization of pH, the group added with 1.25g EDTA, 1.42g Tris and 1.00g Citrate well maintained the neutral range of pH during storage (6.88 at day1 to 7.33 at day6 in Modified Kp), Especially, the concentrations of the buffers added in Modified Kp were lower, until 1/2∼1/4 ranges, than those in Modena and other extenders. The motility of frozen-thawed boar sperm diluted with Modified Kp was significantly higher than that of Basic Kp, BTSg, BTSa and Modena (87.0% vs. 55.0∼71.0% at day1; 13.3% vs. 0∼6.3% at day6), Conclusively, Modified Kp in this experiment was kept the favorable physiological conditions in spite of low concentrations of the buffers and motility of frozen-thawed boar sperm was obtained better than that of Basic Kp and other commercial products such as BTSg, BTSa and Modena.

• Korean Journal of Animal Reproduction
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• v.23 no.3
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• pp.257-262
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• 1999

This study was carried out to evaluate the effect of pH and storage temperature on the viability of boar sperm diluted with extender KpA for swine artificial insemination. The results obtained in this experiment were summarized as follows : 1. The viability of boar sperm diluted with KpA was higher than that with BTS during storage and especially more than 60% of sperm viability in KpA was maintained through 6 days. The pH values of all extenders were kept during storage of semens following dilution. 2. The sperm diluted with acidic (pH 6.3~6.8) or alkalic (pH 7.8~8.0) KpA and stored at 4$^{\circ}C$ or 37$^{\circ}C$ were more sensitive in viability than that with neutral pH (6.8~7.3) and at 17$^{\circ}C$ storage. But pH values of all conditions were not increased rapidly. Especially acidic and alkalic diluents were more stable in pH after dilution. Conclusively, extender pH and storage temperature was the important factors for sperm viability. The KpA setting up around neutral pH was the optimal boar semen extender for maintaining liquid semen at 17$^{\circ}C$.

• Reproductive and Developmental Biology
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• v.34 no.3
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• pp.241-246
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• 2010

The objective of this study was to investigate the effect of storage time on fresh boar semen in Androhep and Beltsville Thawing Solution (BTS). Boar semen samples extended in each extender were stored at $17^{\circ}C$ up to 4 days. Sperm motility kinematics was evaluated by computer assisted sperm analyzer (CASA) and capacitation status by chlortetracycline (CTC)/Hoechst 33258 staining. Sperm motility (%) was not decreased during storage in BTS and Androhep. No significant difference between extenders was observed. Only significant differences in kinematic parameters on linearity during storage were found. The percentage of dead sperm significantly decreased during storage (p<0.05). Also the percentage of noncapacitated, capacitated, and acrosome-reacted sperm significantly modified during storage (p<0.05). However, there was no significant difference between extenders except proportion of capacitated sperm. This finding supported that modification in these parameters was not significantly different between extenders during this short-term storage. Our finding strongly indicated that both Androhep and BIS maintained favorable conditions for motility, motility kinematics, and capacitation status during short-term storage. Despite modifications in some parameters were apparent during sperm storage in extenders, these may not affect the fertilizing capacity of boar semen.

• Reproductive and Developmental Biology
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• v.32 no.1
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• pp.59-64
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• 2008

The objective of this study was to estimate modification of semen quality during storage. Liquid boar semen samples extended in Beltsville Thawing Solution were stored at $17^{\circ}C$ up to 5 days. While % motility and linearity significantly decreased eon day 3 in extender, the qualitative motility patterns were maintained satisfactorily. Also the storage of boar semen up to 5 days before insemination did not significantly changed the acrosome intactness. However, acrosome changed sperm significantly increased and capacitated sperm significantly decreased from day 4. No significant modifications in acrosome integrity were showed during sperm storage; these results suggest that liquid boar semen may keep the quality in extender for 3 days.

• Proceedings of the KSAR Conference
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• 2001.03a
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• pp.77-77
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• 2001

본 연구는 돼지 동결정액의 번식성적을 개선코자 기존의 maxi-straw와 cryogenic-vial을 이용하여 포장방법에 따른 동결방법과 융해방법별 정액성상 및 번식성적을 비교하였다. 동결방법은 두 가지 포장방법 모두 정액의 양(5$m\ell$)과 농도(5.0$\times$$10^{9}$/dose)가 동일한 조건으로 처리하였으며, LYE(Lactose egg york extender) 보존액으로 희석하여 액체질소 상단 15cm에서 20분간 동결하였다. 융해방법은 maxi-straw는 52$^{\circ}C$에서 45초간 cryogenic-vial은 52$^{\circ}C$에서 190초간 융해하여 $25^{\circ}C$로 가온 된 80$m\ell$ BTS (Beltsville thawing solution) 보존액과 혼합하였다. 정액성상검사는 정자자동분석기(SAIS : Sperm Analysis Image System, Korea)를 이용하였다. 총활력(TM : Total motility)과 정자의 빠르기(VCL : Curve linear velocity)는 maxi-straw가 54.3%와 46.6%로 cryogenic-vial의 35.6%와 36.6%보다 우수하였다(P<0.05). 정자의 직진성(STR : Straightness)과 NAR은 maxi-straw가 53.2%와 32.6%로 cryogenic-vial의 47.3%와 29.8%와 비슷한 경향을 나타내었다. 수태율과 분만율 및 총산자수는 maxi-straw가 77.3%, 68.2% 및 8.0두로 조사되어 cryogenic-vial포장방법의 66.7%, 61.9% 및 7.4두보다 다소 우수하였으나 통계적인 유의차는 인정되지 않았다. 이상의 결과로 볼 때 cryogenic-vial방법이 새로운 돼지 동결정액 포장방법의 가능성을 나타낸다고 사료된다.

• Korean Journal of Animal Reproduction
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• v.26 no.1
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• pp.17-23
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• 2002

The present study was carried out to investigate the effects of the maturation media such as a modified TCM-199 (mTCM-199) medium, modified Waymouth MB 752/1 (mWaymouth MB 752/1) medium or NCSU-23 medium on penetrability of pig oocytes by liquid boar sperm. Oocytes (30~40) were transferred into each well of a Nunc 4-well multidish containing 0.5 $m\ell$ maturation medium. When immature pig oocytes were cultured in mTCM-199, mWaymouth MB 752/1 and NCSU-23 maturation media for 44 h in 5% $CO_2$, in air at 38.5$^{\circ}C$, the germinal vesicle breakdown (CVBD) rates of the oocytes were 95.6, 94.1 and 94.9%, respectively, and the maturation rates (metaphase II) of oocytes were 92.5, 90.1 and 91.1%, respectively. No differences were observed among the maturation media. The spermrich portion of ejaculates with greater than 90% motile sperm were used in the experiment. The semen was cooled 22 to 24$^{\circ}C$ over 2 h period. The semen was diluted with Beltsville Thawing Solution (BTS) extender at room temperature to give 2$\times$10$^{8}$ sperm/$m\ell$ in 100 $m\ell$ plastic bottle. Liquid boar semen of 30 $m\ell$ in 100 $m\ell$ plastic bottle was kept at 17$^{\circ}C$ for 5 days. The sperm with greater than 70% motility after day 5 of storage were used for in-vitro fertilization (IVF). After 44 h maturation of immature oocytes, cumulus cells were removed and oocytes (30~40) coincubated far 6 h in 0.5 $m\ell$ mTCM-199 and mTBM fertilization media with 2$\times$1061$m\ell$ sperm concentration. At 6 h after IVF, oocytes were transferred into 0.5 $m\ell$ mTCM-199 and NCSU-23 culture media for further culture 6 or 42 h. Sperm penetration, polyspermy and male pronuclear formation of oocytes at 12 h after IVF, and developmental ability of oocytes at 48 h after IVF were evaluated. The oocytes in combination with NCSU-23 medium for maturation and mTBM medium for IVF increased male pronuclear formation (48.0%) compared to those in combination with mTCM-199 media for maturation and IVF, and mWaymouth MB 752il medium for maturation and mTCM-199 medium far IVF. The rates of cleaved embryos (2~4 cell stage) at 48 h after IVF were 24.1% in combination with mTCM-199 media for maturation, IVF and culture, 43.6% in combination with mWaymouth MB 75211 medium fur maturation and mTCM-199 media for IVF and culture, and 71.2% in combination with NCSU-23 medium for maturation, mTBM medium for IVF and NCSU-23 medium for culture. In conclusion, we found out the oocytes matured in vitro were fertilized by liquid boar sperm stored in BTS extender at 17$^{\circ}C$ for 5 days. We recommend the simple defined NCSU-23 medium for nuclear maturation, mTBM medium and liquid boar sperm for IVF, and NCSU-23 medium for embryo culture.