• Journal of Animal Reproduction and Biotechnology
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• v.34 no.3
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• pp.190-196
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• 2019

Tight junctions are constituents of the blood-epididymis barrier that play roles in regulating the unidirectional transcellular transport of ions, water, and solutes to maintain optimal conditions for sperm maturation and storage. Claudin 1 (Cldn1) and 4 (Cldn4) are known as tight junction proteins and are expressed in the basolateral membranes as well as tight junctions in the epididymis of rodents. Here, we examined the expression and localization of Cldn1 and 4 to determine the function of these proteins in the pig epididymis. Cldn1 was highly expressed in the basolateral membrane of epithelial cells in the caput and corpus regions of the epididymis. In the cauda region, however, Cldn1 labeling was significantly decreased in the basolateral membrane of epithelial cells. In contrast, labeling indicated that Cldn4 was expressed in the basolateral membrane in the cauda region of the epididymis and was present at punctate reactive sites in the caput and corpus regions. However, in no region of the epididymis did we detect colocalization of Cldn1 and 4 with labeled ZO-1, the distribution of which is restricted to the tight junctions. Our results indicate that Cldn1 and 4 were region-specifically expressed in the pig epididymis but not present in the tight junctions of epididymal epithelium. In addition, reciprocal regulation in specific regions of the epididymis between Cldn1 and 4 may play an important role in generating an optimal luminal environment for sperm maturation and storage in the pig epididymis.

• Korean Journal of Agricultural Science
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• v.46 no.1
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• pp.195-204
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• 2019

In this study, we examined the effect of a liposome-based extender (Optixcell) and a tris-citric egg-yolk extender (Triladyl) on the frozen-thawed spermatozoa characteristics and the calving rate. The percentages for the total motility of the frozen-thawed spermatozoa were similar in the Optixcell and Triladyl groups. However, among the motile spermatozoa with a straight line velocity (VSL) ${\geq}25{\mu}m/sec$, the curvilinear velocity (VCL, ${\mu}m/sec$), VSL (${\mu}m/sec$), average path velocity (VAP, ${\mu}m/sec$), amplitude of lateral head displacement (ALH, ${\mu}m$), beat cross frequency (BCF, Hz), and plasma membrane integrity of the frozen-thawed spermatozoa for the Optixcell group were significantly higher than those for the Triladyl group. Furthermore, the calving rate in the Optixcell group (79.0%) was higher than that of the Triladyl group (62.8%). However, the acrosomal membrane integrity of the frozen-thawed spermatozoa in the Optixcell and Triladyl groups was not significantly different. These results indicate that semen freezing with Optixcell improved the motility and plasma membrane integrity of frozen-thawed spermatozoa and the calving rate of Hanwoo cows (native Korean cattle). In conclusion, our results suggest that semen freezing with the liposome-based extender Optixcell is more efficient than with the tris-citric egg-yolk extender Triladyl for improved offspring production.

• Journal of Embryo Transfer
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• v.29 no.2
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• pp.163-169
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• 2014

The objective of this study was to evaluate the characteristics of Korean Native Cattle sperm frozen-thawed with L-cysteine and/or catalase. The semen from bulls was collected by the artificial vagina method, and Triladyl containing 20% egg-yolk and/or L-cysteine (L), catalase (C) and L-cysteine + catalase was added to the diluted semen for cryopreservation. The results showed that sperm viability was significantly higher in the L-cysteine + catalase ($69.49{\pm}3.16%$) group than in the control ($60.5{\pm}3.94%$) group (p<0.05). Acrosome damage was significantly lower in the L-cysteine ($17.12{\pm}1.08%$) group than in the control ($21.46{\pm}1.14%$), catalase ($20.54{\pm}0.76%$), and L-cysteine + catalase ($19.29{\pm}0.65%$) groups (p<0.05). In addition, the level of intact mitochondria in the spermatozoa was significantly higher in the L-cysteine ($58.65{\pm}1.39%$) group than in the control ($50.63{\pm}2.37%$) group (p<0.05). The hydrogen peroxide level in the frozen-thawed sperm was significantly lower in the L-cysteine ($3.74{\pm}1.66%$), catalase ($4.65{\pm}1.87%$), and L-cysteine + catalase ($8.11{\pm}2.15%$) groups than in the control ($13.22{\pm}1.6%$) group (p<0.05). The glutathione level was significantly higher in the L-cysteine ($1.33{\pm}0.03%$) group than in the control ($1.08{\pm}0.06%$), catalase ($1.05{\pm}0.02%$) and L-cysteine + catalase ($1.11{\pm}0.03%$) groups (p<0.05). In conclusion, L-cysteine and catalase could protect the membrane of Korean Native Cattle sperm from damage during sperm cryopreservation. Especially, L-cysteine was more effective for keeping acrosomes and mitochondria intactness during sperm cryopreservation.

• Journal of Embryo Transfer
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• v.29 no.2
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• pp.141-148
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• 2014

Quercetin and genistein, plentifully present in fruits and vegetables, are flavonoid family members that have antioxidative function and plant-derived phytoestrogen activity. The antioxidative effects of quercetin and genistein on boar sperm characteristics and in vitro development of IVF embryo were investigated. The sperm motility was increased by addition of genistein $50{\mu}M$ for 6 hr incubation compared to control (p<0.05). The sperm viability was increased by addition of quercetin 1 and $50{\mu}M$ and genestein 1 and $50{\mu}M$ for 3 hr incubation. In addition, the sperm viability seemed to be increased dose-dependantly by addition of quercetin or genistein 1 and $50{\mu}M$, respectively (p<0.05). The membrane integrities were not increased by quercetin or genistein treatments for 3 hr or 6 hr incubation period except for quercetin $1{\mu}M$ for 3 hr incubation. In mitochondrial activities, addition of quercetin $50{\mu}M$ for 6 hr incubation increased mitochondrial activity but decreased at $100{\mu}M$ concentration compared with control (p<0.05). When porcine IVF embryos were cultured in PZM-3 medium supplemented with low concentrations of quercetin ($1{\sim}10{\mu}M$), the developmental rates to morula and blastocyst increased but significantly decreased at high concentrations of quercetin ($25{\sim}50{\mu}M$). The highest developmental rate to blastocysts among all concentrations of quercetin was shown at quercetin $10{\mu}M$ (p<0.05). The developmental rates to morula or blastocysts at low ($0.01{\sim}1{\mu}M$) and high ($5{\sim}10{\mu}M$) concentrations of genistein were not significantly different among all treatment group and genistein did not affect on IVF embryo development. These results suggest that quercetin and genistein seem to have positive effects at certain concentrations on sperm characteristics such as motility, viability and mitochondrial activity. In addition, low concentrations of quercetin (1, 5 and $10{\mu}M$) in this experiment, seem to have beneficial effect on porcine IVF embryo development but genistein did not affect on it at all given concentrations ($0.01{\sim}10{\mu}M$).

• Journal of Embryo Transfer
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• v.31 no.3
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• pp.287-291
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• 2016

For evaluating the boar semen quality, sperm motility (MOT) is an important parameter because the movement of spermatozoa indicates active metabolism, membrane integrity and fertilizing capacity. Estrogen receptors 2(ESR2) is involved in estrogen related apoptosis in cell cycle spermatogenesis, but their functions have not been confirmed in pig until now. Therefore, this study was conducted to analyze their association with sperm motility and kinematic characteristics. DNA samples from 105 Duroc pigs with records of semen 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 analyzed. A SNP in coding region of ESR2 g.35547A > G in exon 5 was associated with MOT (p < 0.05) in Duroc population. Therefore, we suggest that the porcine ESR2 gene may be used as a molecular marker for Duroc boar semen quality, although its functional effects were not defined yet. These results might shed new light on the roles of ESR2 in spermatogenesis as candidate gene for boar fertility, but still the lack of association across populations should be considered.

• Asian-Australasian Journal of Animal Sciences
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• v.17 no.11
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• pp.1501-1508
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• 2004

In this study the effect of extenders and temperatures on sperm viability and fertilizing capacity of boar sperm during long-term storage was investigated. Acrosomal integrity, membrane integrity, motility and hypo-osmotic resistance were evaluated by fluorescence and light microscopy. An in vitro fertilization test was performed to assess the fertilizing capacity of stored spermatozoa. The five diluents tested were ranked according to their ability to maintain sperm functional parameters and Zorlesco (ZO) extender with BSA or with PVA instead of BSA produced the best results. Zorlesco extender substituted with PVA (ZO+PVA) was found to maintain motility both at 15 and 20$^{\circ}C$. within 5 days of storage, but the quality of semen stored at 15$^{\circ}C$ decreased thereafter as compared to semen stored at 20$^{\circ}C$ Semen stored at 5$^{\circ}C$ demonstrated rapid loss of motility already within 24 h. Both fertilization and cleavage of semen stored at 20$^{\circ}C$ in ZO substituted with PVA instead of BSA did not change significantly until day 8 of storage. It is therefore concluded that PVA can be used to substitute for BSA and 20$^{\circ}C$ was more suitable than 15$^{\circ}C$ for boar semen storage, and in vitro fertilizing capacity of spermatozoa was maintained for at least 8 days in ZO+PVA at 20$^{\circ}C$.

• 대한생식의학회:학술대회논문집
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• 2000.06a
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• pp.13-28
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• 2000

Human spermatozoa exhibit a capacity to generate ROS and initiate peroxidation of the unsaturated fatty acids in the sperm plasma membrane, which plays a key role in the etiology of male infertility. The short half-life and limited diffusion of these molecules is consistent with their physiologic role in key biological events such as acrosome reaction and hyperactivation. The intrinsic reactivity of these metabolites in peroxidative damage induced by ROS, particularly $H_2O_2$ and the superoxide anion, has been proposed as a major cause of defective sperm function in cases of male infertility. The number of antioxidants known to attack different stages of peroxidative damage is growing, and it will be of interest to compare alpha-tocopherol and ascorbic acid with these for their therapeutic potential in vitro and in vivo. Both spermatozoa and leukocytes generate ROS, although leukocytes produce much higher levels. The clinical significance of leukocyte presence in semen is controversial. Seminal plasma confers some protection against ROS damage because it contains enzymes that scavenge ROS, such as catalase and superoxide dismutase. A variety of defense mechanisms comprising a number of antioxidants can be employed to reduce or overcome oxidative stress caused by excessive ROS. Determination of male infertility etiology is important, as it will help us develop effective therapies to overcome excessive ROS generation. ROS can have both beneficial and detrimental effects on the spermatozoa and the balancing between the amounts of ROS produced and the amounts scavenged at any moment will determine whether a given sperm function will be promoted or jeopardized. Accurate assessment of ROS levels and, subsequently, OS is Vital, as this will help clinicians both elucidate the fertility status and identify the subgroups of patients that respond or do not respond to these therapeutic strategies. The overt commercial claims of antioxidant benefits and supplements for fertility purposes must be cautiously looked into, until proper multicentered clinical trials are studied. From the current data it appears that no Single adjuvant will be able to enhance the fertilizing capacity of sperm in infertile men, and a combination of the possible strategies that are not toxic at the dosage used would be a feasible approach.

• Animal Bioscience
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• v.35 no.11
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• pp.1666-1674
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• 2022

Objective: Letrozole, a potent aromatase inhibitor, is known to have the potential to modify male reproductive function by altering sex hormone levels. This study aimed to evaluate the semen and testicular characteristics and hormonal profile of aged Mrakhoz bucks (Capra hircus) treated with letrozole. Methods: Twelve Markhoz male goats, aged between 4.5 to 5.5 years with an average body weight (BW) of 61.05±4.97 kg were used for the study. Animals were randomly divided into two equal groups and subcutaneously received either 0.25 mg/kg BW of letrozole or a control every week for 2 months. The semen collections were performed every 10 days, and blood samples and testicular biometric records were collected at 20 days intervals. Results: Letrozole causes increased testosterone and follicle-stimulating hormone levels, testosterone to estradiol ratio, semen index and reaction time during the period from 20th to 60th days (p<0.05). Furthermore, letrozole-treated bucks had higher semen volume, sperm concentration, and total sperm per ejaculate from 30th to 60th days (p<0.05). However, no differences occurred between the groups in scrotal circumference, relative testicular volume, semen pH, abnormality, acrosome integrity, and membrane integrity of sperm during the study (p>0.05). The serum luteinizing hormone levels, sperm viability, motility, and progressive motility increased, and estradiol levels decreased after 40th to 60th days of letrozole treatment (p<0.05). Conclusion: Letrozole application to aged Markhoz bucks provokes reproductive hormonal axis which, in turn, induces enhancement of semen production and quality.

• Applied Microscopy
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• v.31 no.3
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• pp.223-233
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• 2001

The spermiogenesis of a Korean octopus, Octopus minor, inhabiting western of Korea Sea was observed by electron microscopy . The obtained results are as follows: The spermiogenesis of Octopus miner proceeds through four stages; early- , mid- , and late-spermatid, and mature sperm. An early spermatid is a spherical cell looking light due to the low electron density. The acrosome formed from Golgi complex of the upper nucleus looks dark due to the high electron density. The extra-nuclear rod (enr) stemming from proximal centriole is transformed from round shape into oval shape, elongating to the upper nucleus. In our observation, the axoneme was being formed from distal centriole, and the manchette composed of a number of microtubules is also found around nuclear membrane. In a mid-spermatid, chromatins in the nucleus contract shaping fine threads, and the manchette is also observed around nuclear membrane. Especially, the spherical acrosome is transformed into long oval one which is tinged with a number of horizontal stripes and has the middle electron density. In a late-spermatid, chromatins in the nucleus contract thick and short. Furthermore, the mitochondrial sleeve, in which the axoneme is surrounded with mitochondria, is observed at middle piece. The axoneme has a typical structure of 9+2 and around it, 9 coarse fibers are observed. Also in the acrosome cavity of mature sperm, horizontal striation is found. However, regularly spaced processes are peculiarly observed in there. A sperm is about 390 fm long, whose head is bent a little like a banana while the acrosome region is helical. In the middle piece of sperm, $11\sim12$ mitochondria are surrounding coarse fibers that reach the main piece of tail, while nothing but 9+2 structured axoneme is found in the end piece.

• Journal of Animal Science and Technology
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• v.63 no.1
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• pp.58-68
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• 2021

Several herbs including Artemisia are known to possess conceptive property. In the present study, mouse spermatozoa were incubated with ethanol extract of Artemisia vulgaris leaves. The effect of extract on acrosome exocytosis was studied by labeling spermatozoa with fluorescein isothiocyanate (FITC) peanut agglutinin and by staining with Coomassie blue. Viability and membrane integrity were studied by Trypan-blue staining and hypo-osmotic swelling test. Artemisia extract at very low concentration caused precocious acrosome reaction and loss of sperm viability. Acrosome reaction increased remarkably from 22.63% to 88.42% with increasing extract concentration from 0 to 2,000 ㎍/mL. However, the viability loss of spermatozoa was increased from 11.71% in control to 63.73% in samples treated, evaluated by Trypan-blue staining method. Membrane damage caused by the extract, evaluated by hypo-osmotic swelling test was even low, ranging from 2.27% to only 24.23%. These results indicate that Artemisia extract might block fertilization by causing precocious acrosome exocytosis in spermatozoa. A direct contraceptive effect was tested by injecting the plant extract into the vagina of female mice and then allowing them to mate with normal males. The treated female mice delivered significantly fewer litters in comparison to the control.