• Asian-Australasian Journal of Animal Sciences
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• 제21권4호
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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.

• 한국수정란이식학회지
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• 제27권1호
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• pp.21-28
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• 2012

A study was conducted on four crossbred bulls, used as artificial insemination (AI) sires, to correlate their semen quality with their non return rate (NRR). Semen was collected once a week via an artificial vagina, diluted in egg yolk-citrate and maintained at $+7^{\circ}C$ for three days. It was evaluated for sperm motility, viability, morphology immediately after collection and was examined daily for sperm motility, viability and morphology of acrosome, mid piece and tail for a total of three days. A total of 2016 cows were inseminated by two AI technicians. The proportions of sperm with normal heads were 83.4% (63.7~91.7%), the proportion of spermatozoa exhibiting normal morphology (acrosome, mid piece and tail), motility and viability were 89.2% (82.3~92.0%), 71.3% (61.7~75.0%) and 76.7% (65.7~85.0%), respectively in fresh ejaculates. Sperm motility and sperm viability was significantly ($p$ <0.05) lower in Holstein-Friesian ${\times}$ Local bull than in other bulls during all three days of storage. The overall NRR for four bulls was 82.7% (72.9-87.5%). Bulls with higher sperm motility, viability and normal morphology of spermatozoa of individual bull had significantly (each $p$ <0.05) higher NRR. The highest ($p$ <0.01) NRR (87.5%) was observed in a Red Chittagong bull whose semen qualities were significantly ($p$ <0.05) higher than Holstein-Friesian ${\times}$ Local bull (NNR 72.9%). The results of the present study concluded that NRR at 56 days post AI is related to parameters of semen quality. Therefore, semen evaluation may allow the discarding of bulls with poor fertility in an AI program.

• 한국수정란이식학회지
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• 제28권2호
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• pp.121-125
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• 2013

This study was carried out to investigate the general characteristics of semen such as semen volume, pH, sperm motility and sperm concentration of the semen collected from Shih Tzu dogs (age of 24 to 48 months, weight of 4 to 8 kg) by using the method of digital manipulation of the penis. The effect of preservation temperature and time on motility of fresh semen was also investigated in the present study. Semen was collected for 16 times from 4 male Shih Tzu dogs by multiple ejaculations (four times ejaculation per dog). The average of semen volume, semen pH, sperm motility and sperm concentration of the second fraction containing small volume of the initial third fraction per ejaculation were $2.11{\pm}0.31$ ml, $6.25{\pm}0.07$, $97.59{\pm}1.03%$ and $2.05{\pm}0.14{\times}10^8$ cells/ml, respectively. Average semen volume per ejaculate, semen pH, sperm motility and sperm concentration of the first fraction from the ejaculation were $1.12{\pm}0.15$ ml, $5.99{\pm}0.14$, $16.09{\pm}6.18%$ and $5.16{\pm}2.03{\times}10^5$ cells/ml, respectively. Those of second fraction were $2.07{\pm}0.29$ ml, $6.36{\pm}0.13$, $97.31{\pm}1.36%$ and $2.15{\pm}0.30{\times}10^8$ cells/ml, respectively. Those of third fraction were $2.60{\pm}0.29$ ml, $6.63{\pm}0.08$, $95.72{\pm}1.61%$ and $6.03{\pm}1.83{\times}10^7$ cells/ml, respectively. Sperm motility was significantly higher at $17^{\circ}C$ preservation temperature than at $5^{\circ}C$ or $36^{\circ}C$ during preservation period except 1 h preservation (P<0.05). When preservation temperature was $17^{\circ}C$, sperm motility was $96.69{\pm}1.49%$ at 1 h, $91.38{\pm}1.90%$ at 6 h, $88.38{\pm}2.34%$ at 12 h, $78.13{\pm}4.58%$ at 18 h, $58.44{\pm}8.57%$ at 24 h and $29.56{\pm}5.06%$ at 30 h, respectively.

• Reproductive and Developmental Biology
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• 제34권3호
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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.

• Animal Bioscience
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• 제36권2호
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• pp.209-217
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• 2023

Objective: The conservation of Bali bulls, the Indonesian native breed of cattle, is crucial for cattle breeding in Indonesia. To guarantee the spread of Bali bulls through artificial insemination the quality of the frozen semen must be high. To this end, adding an extender material to semen that increases spermatozoa's survival during cryopreservation is important. Green tea extract (GTE) can be used as cryoprotectant because its high antioxidant activity can help avoid reactive oxygen species formation. Methods: Semen of five Bali bulls from the National Artificial Insemination Center at Singosari, Indonesia was collected routinely twice a week. First, fresh semen inspection was performed to determine the feasibility of using Bali bulls as animal samples. The extender used in this study was Tris-based egg yolk. The samples were divided into four treatments: T0, no GTE added to the extender; T1, 0.05 mg GTE plus 100 mL extender; T2, 0.10 mg GTE plus 100 mL extender; and T3, 0.15 mg GTE plus 100 mL extender. The semen freezing process was conducted according to standard procedures and sperm quality parameters, i.e., sperm motility, viability, abnormalities, and membrane integrity observed pre-freezing and post-thawing. Results: There were significant differences in total motility, progressive motility, viability, and integrity membrane of Bali bull sperm at both pre-freezing and post-thawing after adding GTE into the extender. In contrast, there were no differences in abnormalities among treatments. Conclusion: Adding GTE at a 0.15 mg into 100 mL Tris-based egg yolk extender can improve the quality of cryopreserved Bali bull sperm.

• 생명과학회지
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• 제29권4호
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• pp.395-401
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• 2019

Phthalate는 내분비 교란물질로 호르몬의 변화, 에스토젠, 앤드로젠, 갑상선 호르몬의 분비를 방해한다. 또한, 인간과 동물에서 심혈관질환, 대사작용, 면역 및 번식체계에 영향을 끼친다. Curcumin은 항산화물질로 항염증 활성 및 항암작용에 영향을 미치는 것으로 알려져 있다. 본 연구는 phthalate가 정자의 운동성, 생존율, 미토콘드리아 활성 및 세포막 기능에 미치는 영향을 알아보고자 실시하였다. 또한, curcumin을 처리하여 phthalate에 노출된 정자에 미치는 영향을 분석하였다. 정자의 운동성과 생존율은 di-n-butyl phthalate (DBP), mono-n-butyl phthalate (MBP) 및 di-2-ethylhexyl phthalate (DEHP)을 처리하였을 때 감소하였다(p<0.05). Phthalate는 정자의 미토콘드리아 활성 및 세포막의 기능을 감소시켰다(p<0.05). 그러나, 정자의 운동성과 생존율은 curcumin을 처리하지 않은 것보다 처리한 정자에서 높게 나타났으며(p<0.05), 정자의 미토콘드리아 활성 및 세포막 기능에서도 높게 나타났다(p<0.05). 결론적으로, phthalate는 정자의 생존율과 세포의 기능에 영향을 미칠 수 있고, 이로부터 세포의 기능을 보호하기 위해서는 curcumin의 처리가 필요 할 것으로 생각된다.

• Reproductive and Developmental Biology
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• 제33권1호
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• pp.25-28
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• 2009

This study was carried out to investigate the general characteristics, such as volume, pH, sperm motility and sperm concentration of the semen collected from Beagle dogs (age $24{\sim}48$ months, weight $10{\sim}15\;kg$) by using the method of digital manipulation of the penis, and the effect of preservation temperature and time on motility of fresh semen. Multiple ejaculates were collected from 4 male Beagles. The average volume, pH, motility and sperm concentration of the second fraction (contained with small volume of the third fraction) per ejaculation were $2.94{\pm}0.24(SD)\;ml$, $6.43{\pm}0.42(SD)$, $97.04{\pm}3.50(SD)%$ and $1.67{\pm}0.23(SD){\times}10^8\;cells/ml$, respectively. Average semen volume per ejaculate, semen pH, sperm motility and sperm concentration of the first fraction from the ejaculate were $1.24{\pm}0.20(SD)\;ml$, $6.03{\pm}0.26(SD)$, $1l.30{\pm}4.02(SD)%$ and $7.25{\pm}1.02(SD){\times}10^5\;cells/ml$. Those of second fraction were $2.52{\pm}0.32(SD)\;ml$, $6.32{\pm}0.31(SD)$, $96.25{\pm}3.52(SD)%$ and $2.35{\pm}0.35(SD){\times}10^8\;cells/ml$. Those of third fraction were $2.71{\pm}0.27\;(SD)\;ml$, $6.52{\pm}0.20(SD)$, $95.65{\pm}2.78(SD)%$ and $5.72{\pm}0.29(SD){\times}10^7\;cells/ml$. Motility of semen was higher at $17^{\circ}C$ preservation temperature than $5^{\circ}C$ or $36^{\circ}C$ during preservation period. When preservation temperature was $17^{\circ}C$, motility was $96.54{\pm}2.05(SD)%$ at 1 h, $90.20{\pm}3.90(SD)%$ at 6 h, $89.05{\pm}2.01(SD)%$ at 12 h, $78.21{\pm}3.50(SD)%$ at 18 h, $45.24{\pm}6.25\;(SD)%$ at 24 h and $30.75{\pm}17.24(SD)%$ at 30 h, respectively.

• 한국가축번식학회지
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• 제15권1호
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• pp.1-6
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• 1991

본 實驗에서는 Ca, BSA, heparin과, 精液의 體外貯藏, 및 수소개체가 新鮮精子 및 卵乳液에 부유되어 5$^{\circ}C$에서 4시간 貯藏된 貯藏精子의 活力과 尖帽反應에 미치는 影響을 조사하였던 바, 그 結果는 다음과 같다. 1. Ca, BSA, Ca + BSA, heparin, heparin + Ca, heparin + BSA 및 heparin + Ca + BSA가 첨가된 SCS에서 15분간 培養한 精子의 活力은 처리간에 유의차가 인정되었으며, BSA가 다른 처리보다 유의하게 높았다. 한편 精子의 尖帽反應率은 처리간에 유의차가 인정되었으며, BSA와 Ca + BSA가 다른 처리보다 유의하게 높았다. 2. 精子의 活力은 新鮮精液과 貯藏精液 공히 KNC 1이 KNC 2, HOL 1과 2보다 유의하게 낮았으나, 尖帽反應率은 KNC 1이 KNC 2, HOL 1과 2보다 유의하게 높았다. 즉 精子의 活力과 尖帽反應率은 공히 수소개체간에 차이가 있었다. 3. KNC 1과 KNC 2의 新鮮精子를 SCS, SCS + Ca, SCS + BSA, SCS + Ca + BSA, SCS + heparin, SCS + heparin + Ca, SCS + heparin + BSA 및 SCS + heparin + Ca + BSA 용액에 각각 15분간 培養하였을 때, 精子의 活力은 수소개체간에 유의한 차이가 있었는데, KNC 1에서는 BSA가 다른 처리보다 유의하게 높았으나, KNC 2에서는 BSA, Ca + BSA 및 Ca이 다른 처리보다 유의하게 높았다. 한편 精子의 尖帽反應率은 역시 수소개체간에 유의한 차이가 있었으며, KNC 1에서는 Ca이, KNC 2에서는 Ca + BSA가 다른 처리보다 높았다.

• Reproductive and Developmental Biology
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• 제29권3호
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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.

• Clinical and Experimental Reproductive Medicine
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• 제29권2호
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• pp.149-157
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• 2002

Objectives: To assess the scavenging effect of carnitine derivatives on oxidative damage to sperm during sperm processing, cryopreservation and thawing. Materials and Methods: Fresh semen samples from 20 normal healthy volunteers were collected by masturbation after at least 48 hours abstinence. After liquefaction of semen samples at room temperature, the specimens were diluted with sperm wash media (Ham's F-10, Life technologics) to a uniform density of $20{\times}10^6/ml$. L-carnitine or acetylcarnitine were added with various concentration of $0{\mu}M$, $10{\mu}M$, $30{\mu}M$ in semen sample or cryoprotectant. All specimens were cryopreservated at $-196^{circ}C$ $LN_2$ for 3 days. Sperm motility, vitality, fertilizing capacity, reactive oxygen species formation and the level of lipid peroxidation were analyzed by computer assisted semen analyzer, eosin-nigrosin stain, hypoosmotic swelling test, chemiluminescence and thiobarbituric acid method, respectively, during sperm processing, cryopreservation and thawing. Results: The sperm motility was only increased in proportion to the concentration of acetylcarnitine with no statistical significance (p>0.05). The sperm vitality was also significantly improved in proportion to the concentration of acetylcarnitine with statistical significance (p<0.05). The sperm fertilizing capacity was significantly increased in proportion to the concentration of L-carnitine and acetylcarnitine and reactive oxygen species generation and lipid peroxidation were significantly decreased with same fashion (p<0.05). On comparison of effects between L-carnitine and acetylcarnitine, acetylcarnitine was superior to L-carnitine on the improvement of sperm motility and vitality as well as the suppression of reactive oxygen species generation and lipid peroxidation. Conclusions: These results suggest that carnitine derivatives have a scavenging effect against oxidative damages during sperm processing, cryopreservation and thawing. Therefore, carnitine derivatives may be useful as an oral antioxidant in patients with male infertility due to increased ROS generation.