• 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.

• Journal of Embryo Transfer
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• v.27 no.1
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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.

• Journal of Embryo Transfer
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• v.28 no.2
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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.

• Fisheries and Aquatic Sciences
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• v.27 no.5
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• pp.306-313
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• 2024

The genus Chirostoma is endemic from the Mesa Central of Mexico. It is conformed of 18 species and six subspecies. Five species are in some category of risk, because of this, Chirostoma jordani is an excellent model species to implement biotechnologies like gametes cryopreservation. Aim of present study was to evaluate fertilizing capacity of cryopreserved C. jordani sperm, as alternative to conservation and assisted reproduction in this specie and genus. Males and females were collected from wild Atlangatepec dam stock, Tlaxcala State, Mexico. Seminal quality was evaluated in fresh and cryopreserved semen with three cryoprotective agents (CPAs): 10% dimethyl sulfoxide (DMSO), 10% methanol (MeOH), 14% ethylene glycol (EG) and it was determined its post-thaw fertilizing capacity. Sperm motility percentage decreased during cryopreservation process (p < 0.05). There were not significant differences in post-thaw motility percentage between EG (53.5 ± 1.9%) and MeOH (53.3 ± 1.3%), but DMSO (50.3 ± 0.5%) was significantly different (p < 0.05). Results showed that 0.2 μL fresh semen were enough to fertilize 100% oocytes (n = 60). 10 μL DMSO and 5 μL MeOH and EG cryopreserved semen were necessary to fertilize oocytes 100% (n = 60) (p < 0.05). Cryopreservation and fertilization protocol for C. jordani sperm was efficient and it could be used for its assisted reproduction.

• 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.

• Animal Bioscience
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• v.36 no.2
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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.

• Journal of Life Science
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• v.29 no.4
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• pp.395-401
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• 2019

Phthalate is a chemical endocrine disrupter and interfere with the action of hormones, estrogens, androgens and thyroid hormones. It also affect cardiovascular, metabolic, immune and reproductive system in the human and animals. Curcumin is antioxidant, anti-inflammatory activity and -cancer properties in the human. We studied whether phthalates damage viability, mitochondrial activity and membrane integrity of sperm in boar semen. We also treated curcumin with/without phthalates in the boar semen. Fresh boar semen was treated with phthalates and/or curcumin for examining sperm characteristics. Sperm characteristics, sperm motility, viability, mitochondrial activity, and membrane integrity were determined during storage of boar semen. Sperm motility and viability in dose-dependent manner decreased by di-n-butyl phthalate (DBP), mono-n-butyl phthalate (MBP) and di-2-ethylhexyl phthalate (DEHP, p<0.05). Phthalates also decreased mitochondrial activity and membrane integrity of sperm (p<0.05). However, sperm motility and viability were higher than untreated-curcumin when DBP, MBP and DEHP treated with a curcumin in boar semen (p<0.05). Mitochondrial activity and membrane integrity of sperm were higher in DBP- and MBP-treated semen with curcumin (p<0.05). In conclusion, phthalates can damage sperm viability and quality during the boar semen storage, and curcumin may protect the boar sperms from phthalates during storage term.

• Reproductive and Developmental Biology
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• v.33 no.1
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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.

• Korean Journal of Animal Reproduction
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• v.15 no.1
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• pp.1-6
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• 1991

This study was carried out to investigate the effects of Ca, BSA, heparin, semen storage and individual bull on motility and acrosome reaction of bovine fresh sperm and sperm stored in lactose-egg yolk solution(LES) at 5$^{\circ}C$ for 4hours, and the results obtained were as follows: 1. When sperm was incubated in SCS containing Ca, BSA, Ca + BSA, heparin, heparin + Ca, heparin + BSA, and heparin + Ca + BSA for 15 minutes, there was significant difference in sperm motility among the treatments, especially BSA showed significantly higher sperm motility than the others. Also there was significant difference in sperm acrosome reaction among the treatments, especially BSA and Ca + BSA showed significantly higher sperm acrosome reaction than the others. 2. Bull KNC 1 showed significantly higher sperm motility than KNC 1, HOL 1 and 2 in both fresh and stored semen, however KNC 1 showed significantly lower sperm acrosome reaction than KNC 1, HOL 1 and 2. Therefore, there was significant difference in sperm motility and acrosome reaction among individual bulls. 3. When KNC 1 and KNC 2 sperm were incubated in SCS and SCS + Ca, SCS + BSA, SCS + Ca + BSA, SCS + heparin, SCS + heparin + Ca, SCS + heparin + BSA, and SCS + heparin + Ca + BSA, there was significant difference in sperm motility among individual bulls, especially BSA in KNC 1 and BSA, Ca and Ca + BSA in KNC 2 showedsignificantly higher motility than the others. However, there was significant difference in sperm acrosome reaction among individual bulls, Ca in KNC 1 and Ca + BSA in KNC 2 showed higher acrosome reaction than the others.

• 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.