• Clinical and Experimental Reproductive Medicine
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• v.38 no.2
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• pp.82-86
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• 2011

Objective: To evaluate sperm nuclear DNA fragmentation and chromatin structure after 18 hours' incubation at room temperature. Methods: Twenty-eight male partners who participating IVF treatment were prospectively included in this study. Ejaculated sperm count and motility were assessed. The sperm was then immediately processed by the conventional swim-up method. After utilization of some of the sample for routine clinical use, the remainder of each of the samples was divided into two aliquots. One aliquot was immediately assessed for sperm nuclear DNA fragmentation (TUNEL assay) and chromatin structure (toluidine blue [TB] staining). The other aliquot was incubated at room temperature for 18 hours and then assessed by two methods. Only dark-TB sperms were considered as having abnormal chromatin structure. Data before and after extended incubation were compared using a paired Student's $t$-test. Results: Before and after extended culture, nuclear DNA fragmentation assessed by TUNEL was $4.9{\pm}4.7%$ and $7.0{\pm}6.4%$, respectively ($p$=0.008). The proportion of abnormal chromatin structure (dark-TB sperm) was $8.2{\pm}5.6%$ and $10.3{\pm}6.5%$ ($p$ <0.001), before and after incubation, respectively. Conclusion: After 18 hours' incubation at room temperature, sperm nuclear DNA and chromatin structure were significantly affected. The IVF practitioner should bear this information in mind when performing delayed insemination, especially for $in$ $vitro$ maturation cycles.

• Clinical and Experimental Reproductive Medicine
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• v.16 no.2
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• pp.113-118
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• 1989

In order to study the effects of $Ca^{++}$ and dbcAMP on sperm motility, cauda epididyal spermatozoa of mouse were incubated in the media containing the various concentrations of $Ca^{++}$ and dbcAMP, and Its motility were observed and analyzed. The rate(%) of the sperm with forward motility which may be fertile, reached at the equilibrium state in 30-60 min. after the beginning of incubation. $Ca^{++}$ requirement for the maintenance of sperm motility obviously appeared in 3hrs. after the beginning of incubation. In the medium containing 0.01mM $Ca^{++}$, the rate of sperm forward motility was highest(30.6-40.6%), whereas in 10mM $Ca^{++}$ medium, the rate was rather reduced because of a severe agglutination phenomenon. On the other hand, 1mM dbcAMP in the medium was significantly effective for the maintenance of sperm forward motility.

• Korean Journal of Animal Reproduction
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• v.25 no.4
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• pp.327-337
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• 2001

This study investigated the effects of superoxide dismutase (SOD) on lipid peroxidation and fertilizing ability in vitro of boar spermatozoa frozen-thawed. The percentages of motile sperm were highest when SOD of 10 units/$m\ell$ was added to washing medium for spermatozoa. However, the rates of motile sperm were not significantly different in different concentrations of SOD. On the other hand, the motile rates of sperm washed with SOD were lower in sperm inculbated for 120 min than 30 min regardless of the different concentrations of SOD. The percentage of spermatozoa that reached acrosome reaction were increased with incubation periods prolonged. No significant differences, however, were observed in acrosome reaction rates between sperm incubated with and without SOD supplementation for 0, 60 and 120 min. When oocyies inseminated with different concentrations of SOD, the penetration rates were significantly (P＜0.05) higher in medium with 1 unit/$m\ell$ than 0, 10 and 100 units/$m\ell$ of SOD. However, the proportions of polyspermit oocytes were significantly (P＜0.05) lower in medium with 10 and 100 units/$m\ell$ than 0 unit/$m\ell$ of SOD. In another experiment, the sperm suspension were also treated with different concentrations of SOD and were assayed far sulfhydryl(-SH) group content. In the groups treated with 100 units/$m\ell$ of SOD, sperm-SH group were higher than another groups. However, sperm-SH group content were not siginificantly different in spermatozoa treated with different concentrations of SOD. Under the same conditions, the lipid peroxidation of sperm was evaluated on the basis of malondialdehyde production. The addition of SOD to sperm suspension decreased the formation or malondialdehyde. However, there were not significantly different in sperm treated with different concentrations of SOD. The activity of sperm binding to zona pellucida was also evaluated through binding to salt-stored porcine oocytes. The sperm binding to zona pellucida were gradually increased with SOD concentrations added. The number of spermatozoa binded to zona pellucida were significantly (P＜0.05) higher in medium with 100 units/$m\ell$ than 0 units/$m\ell$ of SOD. These findings suggested that SOD cause an enhancement penetrarion ability and sperm zona binding in boar spermatozoa frozen-thawed.

• Asian-Australasian Journal of Animal Sciences
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• v.19 no.11
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• pp.1561-1565
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• 2006

The objective of this study was to investigate the anti-oxidative effects of taurine on sperm characteristics for in vitro storage of boar semen. Semen was randomly divided into 10 groups in conical tubes and treated with different concentrations of taurine (25-100 mM) with or without $250{\mu}M$ $H_2O_2$. The percentage of motile spermatozoa in taurine groups after 6 and 9 h were significantly higher at >94% and 87%, respectively, compared to the control group ($85.1{\pm}0.5$ and $72.4{\pm}0.3$, p<0.05). The sperm motility in taurine with $H_2O_2$ after 6 h incubation was slightly decreased compared to the taurine alone treatment, but after 9 and 12 h incubation % sperm motility dropped sharply in taurine with $H_2O_2$ ($75.3{\pm}0.3$ and $69.6{\pm}2.9$, p<0.05). For 3, 9 and 12 h incubation, sperm viability in the control was lower than in taurine groups, irrespective of taurine concentration. In eosin Y and nigrosin staining (ENS), the sperm survival rates (%) for 6 h incubation were significantly higher in 25 mM ($76.0{\pm}0.6$) and 50 mM taurine groups ($78.0{\pm}0.7$), respectively. Sperm survival rates for 9 and 12 h incubation were higher in taurine groups (${\geq}48%$ in 9 h and ${\geq}42%$ in 12 h) compared to controls ($43.0{\pm}2.1$ and $31.0{\pm}0.6$, respectively). In the hyoosmotic swelling test (HOST), sperm membrane integrity was similar to the results of sperm survival. These experiments indicate that supplementation of taurine to the semen extender can increase the sperm characteristics(motility, viability, survival and membrane integrity).

• Korean Journal of Animal Reproduction
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• v.14 no.1
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• pp.84-91
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• 1990

Experiments were disigned to define and optimize efficiency of a system whereby pig follicular oocytes could be matured and fertil ized in vitro. The pig oocytes removed from 1- 2 mm and 3-7 mm follicles were cultured in vitro in the mKRB(-BSA) solution containing estrous sow serum (ESS), FCS or dialyzed pig follicular fluid for 24 to 48 hr at 37$^{\circ}C$. The oocytes matured in vitro were evaluated after epididymal spermatozoa-oocyte incubation for 24 hr for pronucleus formation. 50-60% of the oocytes reached metaphase II during 36 to 48 hr of culture. There was no differernce in oocyte matura¬tion between two groups of follicular size but meiosis was slightly faster in the 3-7 mm follicular oocytes. The oocytes matured in mKRB (-BSA) plus 5% ESS, 15% FCS or dialyzed follicular fraction showed slightly higher maturation rates than the control mKRB. in vitro fertilization, pronucleus formation, tended to be increased when mKRBi-BSA) plus 5% ESS or 15% FCS was used for oocyte maturation and in vivo -capacitated spermatozoa were inseminated, respectively. It is concluded that ESS, FCS and dialyzed pig follicular fluid may be effective factors for in vitro maturation and fertilization of pig follicular oocytes.

• Korean Journal of Animal Reproduction
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• v.12 no.2
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• pp.112-119
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• 1988

These studies were conducted to investigate the effects of culture temperature and time on the in vitro maturation and semen type and media on the in vitro fertilization of bovine follicular oocytes, and to asses in vitro fertilization rate of oocytes cultured by extraffollicular method following fertilization in vitro, or transfer into the pseudopregnant rabbit oviduct or uterus. The bovine oocytes recovered from follicles were cultrued for 18 hrs or 72hrs at 38$^{\circ}C$ with 5% CO2 in moist air. Flesh-diluted(2 folds) and frozen-thawed semen in 0.5ml straw from a fertile bull were used. In order to obtain capacitation of spermatozoa were treated with bovine follicular fluids(BFF) and Inophore A(IA). The results obtained were summarized as follows: 1. The oocytes were classified as "A, B, C, D and Degenerative" depending morphological integrity and those were 62.0%, 12.0%, 17.2%, 5.9% and 3.0% of the total oocytes harvested, respectively. 2. The oocytes matured to metaphase II were significantly increased between 24-48hrs of incubation and at 37-39$^{\circ}C$ with 5% CO2 in moist air. 3. The in vitro fertilization rate following transferred into rabbit oviduct or uterus with bull semen and in vitro matured oocytes were higher ligation than non-ligation of oviduct or uterus. 4. The in vitro fertilization rate of oocytes matured in vitro were higher neat than frozen semen and treatment of IA than BFF on the capacitation of spermatozoa. 5. The effects of semen types and media on in vitro fertilization of oocytes matured in vitro were higher fertilization rate of neat than friozen semen, and media was not significant.

• 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 Embryo Transfer
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• v.24 no.2
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• pp.89-95
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• 2009

Successful in vitro embryo production heavily relies on the normal maturation and fertilisation of oocytes. We examined the normal and abnormal fertilisation of zebu cattle oocytes matured in vitro. Immature cumulus oocyte complexes (COCs) from zebu cattle ovaries at slaughter were matured in vitro (IVM) for 24 h. The oocytes were either fixed, stained and examined for nuclear changes or fertilised in vitro (IVF) with Percoll-separated, heparintreated spermatozoa (1.0 ${\times}$ $10^6$/mL) of zebu (n = 7) and crossbred bulls (n = 7). After 18 h of sperm-COCs co-incubation at $39^{\circ}$C with 5% $CO_2$ in humidified air, the presumptive zygotes were fixed, stained and examined for pronuclei. The number of oocytes retrieved per ovary was 5.4 ${\pm}$ 0.7. The percentage of matured oocytes was 73.0. The difference in motility of spermatozoa before and after Percoll seperation was significant (p<0.001). The percentages of normal and abnormal fertilisation (polyspermia and oocytes with one pronucleus) varied significantly depending on individual bulls (p<0.05). A protocol for IVF of IVM oocytes in Bangladeshi zebu cattle is developed. A future study may elucidate the capacity of such IVM-IVF oocytes to develop to the blastocyst stage for transfer to surrogate mother.

• Journal of Life Science
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• v.33 no.7
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• pp.586-592
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• 2023

Oxidative stress is a critical factor affecting the quality and viability of sperm during boar semen storage. Oxidative stress is also a significant concern during the process of freezing semen. The process of semen storage involves exposing the sperm to various stressors, including temperature changes, cryoprotectants, and extended periods of incubation. In addition, oxidative stress can lead to the production of reactive oxygen species (ROS) within the sperm, resulting in oxidative damage to cellular components, such as lipids, proteins, and DNA. Striking a balance between ROS production and the antioxidant defense system is crucial for maintaining sperm viability and functionality during semen storage. Moreover, the prolonged storage of boar semen leads to an increase in ROS levels, which can impair sperm motility, membrane integrity, and DNA integrity. ROS-induced lipid peroxidation affects the fluidity and stability of sperm membranes, leading to decreased sperm motility. Moreover, oxidative damage to the DNA can result in DNA fragmentation, compromising the genetic integrity of the sperm. In conclusion, oxidative stress is a significant challenge in maintaining sperm quality during boar semen storage. Understanding the mechanisms underlying oxidative stress and their impacts on sperm function is crucial for developing effective strategies to minimize oxidative damage and improve sperm storage outcomes.

• Asian-Australasian Journal of Animal Sciences
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• v.21 no.12
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• pp.1721-1727
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• 2008

Four experiments were conducted to study the following: i) the influence of different concentrations of sucrose (0.15, 0.3 and 0.5 M with osmolality of 308, 500 and 760 mOsm/kg, respectively) in diluents and control diluent (370 mOsm/kg) on intensity of motility and progressive motility of goat sperm without rehydration and freezing step in four incubation periods (0, 0.5, 2 and 4 h after dilution); ii) the influence of gradual dilution (in 3 steps) on improvements in ascertained results of the first experiment; iii) cryoprotective effects of different concentrations of sucrose (0.15, 0.22, 0.29 and 0.37 M with osmolality of 450, 560, 740 and 920 mOsm/kg, respectively) plus 7% glycerol and 20% egg yolk in basic diluent (Tris-Citric acid-Fructose) and iv) the effect of two concentrations of sucrose (0.15 and 0.22 M) with and without glycerol (7%). In experiment 1, we obtained better results for control diluent, 0.15 and 0.3 M sucrose supplemented diluents with 0 and 0.5 h incubation periods. In experiment 2, apart from a slight improvement, similar tendencies to experiment 1 were observed. In experiment 3, we obtained the best result for diluent with 0.22 M sucrose with regard to intensity of motility, progressive motility, live sperm and normal acrosomes ($40{\pm}4%$, $3.1{\pm}0.2$, $37{\pm}4%$ and $37{\pm}4%$, repectively). In experiment 4, we obtained the best result for diluent with 0.22 M sucrose plus 7% glycerol in regard to intensity of motility, progressive motility and live sperm ($39{\pm}3%$, $3.6{\pm}0.4$ and $41{\pm}4%$, respectively). The characteristic normal acrosomes in diluents without glycerol, i.e. diluents with 0.15 and 0.22 M sucrose showed better results ($39{\pm}8$ and $42{\pm}6%$ respectively). With regard to the release of hyaluronidase enzyme there were no significant differences between diluents (p>0.05). The results of the diluents with 0.15 and 0.22 M sucrose without glycerol were slightly lower than those with glycerol ($69{\pm}11$ and $70{\pm}11$ vs. $72{\pm}11$ and $70{\pm}11{\times}120{\times}10^6$ units $ml^{-1}$, respectively). In conclusion, the use of concentrated sucrose solutions showed that goat sperm can tolerate osmolality up to 560 mOsm (0.22 M) in the freezing period. In addition, glycerol proved to be a necessary cryoprotective agent in the cryopreservation of goat sperm, particularly for intensity of motility, progressive motility and live sperm.