• Journal of Animal Science and Technology
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• v.55 no.4
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• pp.237-247
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• 2013

This study was to examine single or combined in vitro effects of environmental endocrine disruptors on boar sperm characteristics, oxidative stress damage in sperm and development of porcine IVF embryos. Addition of various concentration of NP (10, 20, $30{\mu}M$), DBP (10, 50, $100{\mu}M$) and BPA (1, 5 or $10{\mu}g/ml$) on boar sperm characteristics such as percentages of sperm motility, viability, membrane integrity and mitochondrial activity were dose-dependently decreased within 3, 6 or 9 hr incubation period (p<0.05). The overall detrimental effects increased with incubation time increasement. NP, DBP and BPA showed the detrimental effects on sperm membrane and mitochondria of energy production organelles affecting cell viability with the dependancy of dose and incubation time. In combination effects, NP ($10{\mu}M$) + DBP ($10{\mu}M$) significantly decreased boar general sperm characteristics for 3 or 6 hr incubation period compared with control (p<0.05). When both of NP and DBP concentrations (NP; $30{\mu}M$, DBP; $100{\mu}M$) increase, the detrimental effects on sperm characteristics were larger than those of low concentration combination (p<0.05). The inhibitory effects of NP ($30{\mu}M$) + BPA ($10{\mu}g/ml$) on sperm characteristics were larger than those of NP ($10{\mu}M$) + BPA ($1{\mu}g/ml$) (p<0.05). DBP ($100{\mu}M$) + BPA ($10{\mu}g/ml$) decreased sperm characteristics compared with the low concentration combination (DBP $10{\mu}M$ + BPA $1{\mu}g/ml$, p<0.05). This result indicates the detrimental effects of both chemicals on sperm characteristics were dose dependent. Addition of NP ($30{\mu}M$) + DBP ($100{\mu}M$), NP ($30{\mu}M$) + BPA ($10{\mu}g/ml$), DBP ($10{\mu}M$) + BPA ($1{\mu}g/ml$) or DBP ($100{\mu}M$) + BPA ($10{\mu}g/ml$) significantly increased lipid peroxidation for 3 or 6 hr incubation period (p<0.05) compared with no addition control. NP (${\geq}20{\mu}M$) decreased the percentages of IVF embryo development from morulae and blastocyst stages (p<0.05) and its detrimental effects were dose-dependant. BPA 0, 1, 5 or $10{\mu}g/ml$ decreased significantly and dose-dependently the percentage of morulae plus and blastocysts (p<0.05). Combinations of DBP ($100{\mu}M$) plus NP ($30{\mu}M$) and DBP ($100{\mu}M$) plus BPA ($10{\mu}g/ml$) did not affect on morulae and blastocyst development, but NP ($30{\mu}M$) plus BPA ($10{\mu}g/ml$) has significant detrimental effect on embryo development at these stages (p<0.05). These overall results indicate that the partial detrimental effects on boar sperm characteristics and embryo development by NP, DBP, BPA or the combination of these chemicals might be due to the increasement of lipid peroxidation and free radical formation in the cell and there were no specific interaction effects on boar sperm and embryo degeneration among the combined treatments.

• Reproductive and Developmental Biology
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• v.31 no.3
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• pp.175-179
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• 2007

The objective of this study was to investigate the effect of thawing temperature on the sperm viability and acrosomal morphology for semen storage of miniature pig by the 0.5ml straw method. In this present study, sperm viability (SYBR-14/PI staining), membrane integrity (Hypoosmotic Swelling Test), acrosome intactness, intensity and capacitation status (chlorotetracycline staining) in frozen miniature pig sperm were evaluated after thawing at 37, 50 and $70^{\circ}C$ for 5, 10 and 45 sec, respectively. Interestingly, the results indicated that sperm thawed at $70^{\circ}C$ for 5 sec significantly (p<0.05) increased sperm viability, but lower the percentage of AR (acrosome reacted spermatozoa) pattern compared to sperm thawed at $37^{\circ}C$ for 45 sec and $50^{\circ}C$ for 10 sec. In terms of thawing condition, high temperature for a short time using the 0.5ml straw was improved cryosurvival of miniature pig semen. Therefore, appropriate thawing method for cryopreservation of miniature pig is required for increasing post-thawing viability.

• Reproductive and Developmental Biology
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• v.35 no.3
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• pp.377-383
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• 2011

The objective of this study was to examine effect of ethylene glycol for semen cryopreservation in Korean Jeju Black Bull. The semen was cryopreserved with extenders containing cryoprotectants (7% glycerol and 3%, 5%, 7% ethylene glycol) and packed to 0.5 ml straws. The semen straws were located above 3 cm of liquid nitrogen for 5 min, 5 cm for 10 min and 8 cm for 10 min. And then frozen straw was plunged into $LN_2$. Post-thawed sperm motility, viability and membrane integrity were significantly higher in 5% ethylene glycol ($72.5{\pm}5.00%$, $54.88{\pm}0.66%$ and $46.00{\pm}2.40%$; p<0.05). Motility and viability were similar between 7% glycerol and 5% ethylene glycol. However, the membrane integrity was significantly higher in 5% ethylene glycol ($34.69{\pm}4.64%$ vs $46.00{\pm}2.40%$; p<0.05). The viability and membrane integrity were significantly higher in 5 cm for 10 min and 8 cm for 10 min than 3 cm for 5 min (viability: $55.81{\pm}2.94$, $55.19{\pm}3.34$ vs $47.94{\pm}3.48%$; p<0.05 and membrane integrity: $44.94{\pm}3.51$, $46.06{\pm}2.25$ vs $40.38{\pm}1.03%$; p<0.05). The percentage of capacitated sperm assessed by CTC staining, percentage of F pattern was higher in 7% glycerol, 5% and 7% ethylene glycerol, and AR pattern was significantly higher in 3% ethylene glycol. F pattern was significantly increased in 5 cm for 10 min and 8 cm for 10 min (p<0.05), but AR pattern was significantly increased in 3 cm for 5 min (p<0.05).

• Journal of Animal Science and Technology
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• v.54 no.2
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• pp.95-101
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• 2012

The objective of this study was to examine the effect of amides as a cryoprotectant for semen cryopreservation in Korean Jeju Black Bull. The semen was cryopreserved with extenders containing 5% dimethyl acetamide (DMA), 5% dimethyl formamide (DMF), 5% methyl formamide (MF) or 7% glycerol. Post-thawed sperm were evaluated for sperm motility, viability, acrosome integrity and membrane integrity. Post-thawed sperm motility was significantly higher (p<0.05) in glycerol and DMF ($64.00%{\pm}9.62$ and $59.00%{\pm}5.48$, respectively) than DMA and MF ($50.00%{\pm}3.24$ and $44.00%{\pm}4.18$, respectively). Sperm viability wassignificantly higher (p<0.05) in glycerol and DMF ($58.25%{\pm}7.35$ and $53.05%{\pm}3.77$, respectively) than others. However, for sperm motility and viability, there were no differences among glycerol and DMF. Also, swelling sperm ratio by hypo-osmetic selling test (HOST) was significantly increased (p<0.05) in glycerol and DMF treatments ($45.12%{\pm}25.08$ and $44.95%{\pm}8.58$, respectively). The percentage of capacitated sperm assessed by CTC staining, F pattern was lower (p<0.05) in DMF than others. B pattern was increased (p<0.05) in DMA, DMF and MF when compared with glycerol. AR pattern ratio was decreased (p<0.05) in glycerol and DMF when compared with DMA and MF. These results suggested that amides performed better and could be used as a cryoprotectant for semen freezing of Korean Jeju Black Bull.

• Asian-Australasian Journal of Animal Sciences
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• v.24 no.10
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• pp.1348-1357
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• 2011

The objective of this study was to compare the effect of Butylated Hydroxy Toluene (BHT), Pentoxifylline (PTX) and ${\alpha}$-tocopherol (Vit E) on semen quality parameters of Karan Fries bulls. The fortification of extender by various semen additives improves motility as well as fertility of spermatozoa. Split samples of 24 ejaculates of four Karan Fries bulls were extended in extender with or without various additives such as BHT, PTX and Vit E, and performance was evaluated at an interval of 0, 24, 48 and 72 h at refrigerated temperature (4-$7^{\circ}C$). Results of the present study revealed that addition of BHT, PTX and Vit E in extender improved sperm cell function, such as motility, viability, HOST, and acrosome integrity, as compared to the control during liquid storage up to 48 h of preservation at refrigerated temperature. There was no significant (p<0.05) difference between any of the additives up to 48 h of preservation. Overall, the results showed a significant (p<0.05) deterioration in motility after each storage interval. The results showed a significant deterioration in the acrosome integrity and plasma membrane integrity up to 48 h; subsequently, there was not much degradation of both the semen quality parameters. There was a significant increase in spermatozoal tail and total abnormality after each storage interval at refrigerator temperature (4 to $7^{\circ}C$); however, the head and mid-piece abnormalities were almost unaffected. Tail and total abnormality were least in extender fortified with BHT, PTX and Vit E at different hours of incubation as compared to the control. The addition of 1.5 mM BHT, 3.6 mM PTX and 1 mg/ml Vit E in the semen extender has more beneficial effect in terms of semen quality and preservability of spermatozoa.

• Journal of Veterinary Clinics
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• v.24 no.4
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• pp.486-492
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• 2007

Seminal plasma(SP) is usually removed from semen that is to be cryopreserved. However, some reports indicate that SP has beneficial effects on spermatozoa during chilling and freezing. The purpose of this study was to determine the effect of SP on sperm survival by adding SP to the extender before cooling and freezing canine spermatozoa. In replicate experiments, ejaculates obtained from four healthy dogs(1-4 years old) of various breeds were pooled, centrifuged at $300{\times}g$ for 10 min at $25^{\circ}C$, and the supernatant of seminal plasma was decanted. Spermatozoa were suspended in egg yolk-Tris(EYT) buffer. The study comprised two experiments: [Exp 1] Sperm were suspended in EYT extender containing either 0, 20, 40, 80 or 100% SP and were slowly cooled to $4^{\circ}C$ for 2h or held at $25^{\circ}C$ as controls. Sperm concentration was adjusted to $2{\times}10^8/ml$. [Exp II] Sperm samples, each of which contained $1{\times}10^8/ml$, were assigned to nine groups to be frozen. In the first four groups, sperm in EYT containing either 20, 40, 80 or 100% SP were cooled to $4^{\circ}C$, then diluted to contain final concentrations of EYT+0.6M glycerol and then were frozen. The final concentrations of SP were 10, 20, 40 or 50%. In the other four groups, sperm in EYT alone were first cooled slowly to $4^{\circ}C$, then diluted to contain final concentrations of EYT+0.6M glycerol plus 10, 20, 40 or 50% SP and then were frozen. Spermatozoa, which chilled in EYT alone and diluted to contain final concentrations of EYT+0.6M glycerol without seminal plasma, and then frozen, was regarded as control. Spermatozoa were frozen at $25^{\circ}C/min$ of cooling rate in plastic straws that were suspended above liquid nitrogen and thawed in water at $38^{\circ}C$ for 1 min. Sperm survival was assayed by determining progressive motility and integrity of plasma and acrosome membranes. Progressive motility was determined by microscopic examination at $200{\times}$ magnification. Membrane integrity was assessed by use of a double fluorescent dye, and acrosome integrity by staining sperm with Pisum sativum agglutinin. The results of the first experiment showed that adding SP did not improve motility of spermatozoa compared to those incubated without SP regardless of temperature. The results of the second experiment showed that spermatozoa suspended in EYT+0.6M glycerol containing SP exhibited the higher progressive motility before being frozen(P<0.05). However, frozen-thawed spermatozoa that had suspended in EYT+0.6M glycerol containing SP showed the similar or lower viability(P<0.05). In summary, although seminal plasma did not affect spermatozoa that were chilled in EYT without cryoprotectant(CPA), addition of seminal plasma to EYT containing CPA did significantly improved progressive motility of canine spermatozoa that were chilled.

• Reproductive and Developmental Biology
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• v.37 no.3
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• pp.155-159
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• 2013

This study was carried out to investigate synthetic extender for semen cryopreservation of Jeju Native Black Bull. The semen was collected using an artificial vagina and transported to the laboratory. The semen was diluted 1:1 by Tris-Egg yolk extender and contrifuged in 1,500 rpm for 15 minutes. The supernatant was removed. The pellect was diluted to final sperm concentration of $2{\times}10^8/ml$ by doubling in every 30 minutes at $4^{\circ}C$ cold chamber. The semen was equilibrated for 4 hours at cold chamber and packed to 0.5 ml straw. The semen straws were located above 5 cm for 10 minutes. The height and duration affect the freezing speed by temperature. The frozen straw was plunged to $LN_2$. The presented straws were examined the viability and motility after thawed at $37^{\circ}C$ water bath. Frozen-thawed sperm were evaluated sperm viability, membrane integrity and acrosome integrity. Post-thawed sperm viability has been significantly higher (p<0.05) in fresh sperm ($93.27{\pm}1.62%$) than frozen-thawed sperm ($73.34{\pm}3.27%$). However, there were no significant differences between fresh and frozen-thawed dead cell rate ($7.35{\pm}2.63$ vs, $13.71{\pm}2.85$). In sperm motility, between Triladyl and AndroMed Extender, there was no significant different ($72.86{\pm}2.83$ vs, $81.47{\pm}2.48$), similarly, the dead cell rates was similar ($18.41{\pm}3.42%$ and $17.26{\pm}4.25$). The results of our study suggest that AndroMed to the freezing extender showed more positive effect on the frozen-thawed spermatozoa in Jeju Native Black bull semen.

• Journal of Animal Science and Technology
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• v.58 no.7
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• pp.25.1-25.6
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• 2016

Background: The current study was designed to investigate the effect of supplementation of Flaxseed (Linumusitatisimum) oil on libido and semen quality of Nilli-Ravi buffalo bulls. Methods: In this study, 12 adult healthy bulls kept at the Semen Production Unit, Qadirabad district Sahiwal, were used. These bulls were divided into three equal groups, A, B and C. Group A was kept as control, while in groups B and C supplementation of feed was provided by using flaxseed oil @125 ml/day and 250 ml/day,respectively for 12 weeks. Two ejaculates per animal were collected at 0 day then 5th, 6th, 7th, 8th, 9th, 10th, 11th and 12th week of treatment. In this way a total 216 samples were taken, and each semen sample was evaluated for color, volume, mass activity, percent motility, sperm cell concentration per ml, percentage of live sperm, and plasma membrane integrity. Libido of bulls was also evaluated before every collection. Results: Analysis of data revealed that these parameters were significantly (P < 0.01) increased in flax oil treated animals as compared to control. Conclusion: It was concluded from the present study that flax seed oil has beneficial effects on reproductive health of buffalo bull.

• Animal Bioscience
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• v.34 no.5
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• pp.844-854
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• 2021

Objective: The potential of extra virgin olive oil (EVOO), betaine (BET), and ginger (GIN), as natural antioxidants, in reducing negative effects of heat stress on physiological responses, antioxidant capacity, semen quality and fertility of bucks under heat stress were investigated. Methods: Forty adult Animal Production Research Institute line rabbit bucks were distributed randomly into four experimental treatments of ten rabbits each. The first treatment was fed the commercial pellet diet (CPD) without supplementation and served as a control. The other three treatments were fed CPD supplemented with EVOO (300 mg), BET (1,000 mg), and GIN (200 mg) per kg diet for 3 consecutive months during the summer season. Results: Supplementation of EVOO, BET, or GIN improved (p<0.05) the sexual desire, progressive motility, vitality, intact acrosome and membrane integrity, sperm cell concentration, sperm outputs and fertility. Seminal plasma total proteins, globulin, total antioxidant capacity, glutathione and glutathione S-transferase, and initial fructose increased (p<0.05), while total lipids, aspartate and alanine aminotransferases and malondialdehyde decreased (p<0.05) compared with the control. In comparing the natural antioxidants treatments, GIN evoked the largest improvement. Conclusion: The inclusion of GIN (200 mg/kg diet) appeared to improve the sexual desire, semen quality and oxidative stress of bucks. This may be a beneficial supplement for the management of rabbit bucks used in natural mating or artificial insemination.

• Journal of Embryo Transfer
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• v.31 no.3
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• pp.293-297
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• 2016

For evaluating the boar semen quality, sperm motility is an important parameter because the movement of sperm indicates active metabolism, membrane integrity and fertilizing capacity. Phospholipase C zeta (PLCz) is important enzyme in spermatogenesis, but the effect has not been confirmed in pigs yet. Therefore, this study was aimed to analyze their association with sperm motility and kinematic characteristics. DNA samples from 124 Duroc pigs with records of sperm motility and kinematic characteristics [total motile spermatozoa (MOT), curvilinear velocity (VCL), straight-line velocity (VSL), the ratio between VSL and VCL (LIN), amplitude of lateral head displacement (ALH)] were subjected. A SNP in non-coding region of PLCz g.158 A > C was associated with MOT (p < 0.05), VCL (p < 0.01), LIN (p < 0.01) and ALH (p < 0.05) in Duroc population. Therefore, we suggest that the intron region of the porcine PLCz gene may be used as a molecular marker for Duroc boar semen quality, although its functional effect was not defined yet. Whether the association is due to the candidate gene or not require further verification. Thus, it will be of interest to continue association studies in the regions surrounding those genes.