• 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.

• Korean Journal of Animal Reproduction
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• v.26 no.3
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• pp.263-273
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• 2002

This study was undertaken to evaluate the effects of $\beta$-mercaptoethanol ($\beta$-ME) on lipid peroxidation and fertilization ability in vitro by xanthine (X) - xanthine oxidase (XO) system in boar spermatozoa frozen-thawed. The boar spermatozoa were treated with X and/or XO, and the spermatozoa viability were measured by the eosin-nigrosin stain method. In control group, level of vitality in boar spermatozoa were higher than in medium with X, XO and X+XO groups. No significant differences, however, were observed under the all conditions. The percentage of spermatozoa that reached acrosome reaction were significantly (P<0.05) higher in sperm treated without that than with $\beta$-ME under the all conditions. On the other hand, when spermatozoa were inseminated in medium with X and/or XO, the penetration rates in all conditions were higher in medium with that than without $\beta$-ME. However, significant differences were not observed between medium with and without $\beta$-ME. The lipid peroxidation of sperm was evaluated on the basis of malondialdehyde (MDA) production. The MDA were higher in sperm treated without that than with $\beta$-ME under the above all conditions. However, significant differences were not observed between medium with and without $\beta$-ME. Sperm-SH group were higher detected in medium with that than without $\beta$-ME under the all conditions. The activity of sperm binding to Bona pellucida was also evaluated through binding to salt-stored porcine oocytes. In control group, sperm binding to zona pellucida were significantly (P<0.05) higher than in medium with X+XO groups. The sperm binding in all conditions were higher in medium with that than without $\beta$-ME. However, significant differences were not observed between medium with and without $\beta$-ME. These results suggest that addition of $\beta$-ME in X-XO system may play a positive role in improving of fertilization ability in vitro.

• Korean Journal of Animal Reproduction
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• v.26 no.3
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• pp.229-234
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• 2002

This study was carried out to investigate the general characteristics such as concentration sperm motility and abnormality of sperm on the whole epididymal semen(EWS), RSP-S(removed seminal plasma by saline) and RSP-T(removed seminal plasma by tris-buffer) semen and survival rates after freezing on motility of whole and RSP-S and RSP-T semen and extender containing 2~8% glycerol, and ability of frozen-thawed sperm to penetrate homologous oocytes. 1. The concentration, motility and abnormality of epididymal WES, RSP-S and RSP-T sperm were 4.25 $\pm$ 0.25($\times$10$^{6}$ Cells/$m\ell$), 3.85$\pm$0.20($\times$10$^{6}$ Cells/$m\ell$), 4.05 $\pm$ 0.28($\times$10$^{6}$ Cells/$m\ell$), 50.55 $\pm$ 2.75%, 67.25 $\pm$ 2.55%, 78.75 $\pm$ 3.55 and 9.45 $\pm$ 2.25%, 37.75 $\pm$ 2.10%, 24.25 $\pm$ 1.55%, respectively. 2. The survival rates of slow and rapid frozen epididymal RSP-S and RSP-T sperm were 35.00 $\pm$ 2.35%, 45.50 $\pm$ 2.15％ and 16.50 $\pm$ 3.55%, 22.55 $\pm$ 3.95%, respectively. The survival rate of epididymal WES and RSP-T sperm after freezing following dilution with tris-buffer containing 2~8% glycerol were 9.25 $\pm$ 1.55%~17.50 $\pm$ 2.50%. 3. The percentage of capacitated and acrosome-reacted sperm prier to culture for fresh and frozen -thawed epididymal RSP-T semen were 45.25 $\pm$ 5.75%, 7.06 $\pm$ 0.25%, 48.20 $\pm$ 6.80% and 13.00 $\pm$ 2.35%, 3.55 $\pm$ 0.85%, 15.50 $\pm$ 1.90%, respectively. The penetration rate the number of sperm per penetrated for fresh and frozen-thawed epididymal RSP-T sperm were 39.25 $\pm$ 4.72%, 34.24 $\pm$ 3.93% and 1.30 $\pm$ 0.33, 1.10 $\pm$ 0.50., respectively.

• 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).

• Reproductive and Developmental Biology
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• v.36 no.3
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• pp.199-206
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• 2012

Cryopreservation induces sublethal damage to the spermatozoa, which leads to their reduced fertile life. This study was designed to determine effect of glycerol and ethylene glycol as cryoprotectant in extender on improve the freezability of Jeju horse semen. The semen was cryopreserved with glucose-EDTA extender containing each 5% glycerol, 5% ethylene glycol, 8% glycerol or 8% ethylene glycol, respectively. Post-thawed sperm were evaluated motility, viability, Membrane integrity and acrosome integrity. Post-thawed sperm motility were not significantly differences among treatments. However, sperm viability were significantly higher (p<0.05) in 8% glycerol ($39.85%{\pm}11.41$) than in 5% glycerol treatment ($18.08%{\pm}1.61$). In membrane integrity, swelling sperm ratio was significantly higher (p<0.05) in 8% glycerol ($34.12%{\pm}11.02$) than other treatments. In the percentage of capacitated sperm assessed by CTC staining, F pattern was significantly higher in 8% ethylene glycol than 5% glycerol and 5% ethylene glycol (p<0.05). B pattern ratio was significantly increased in 5% ethylene glycol compared with 8% glcerol and 8% ethylene glycol (p<0.05). Moreover, 8% ethylene glycol treatment was significantly decreased AR pattern ratio compared with other treatments (p<0.05). It is concluded that treatment of 8% glycerol was improved the sperm viability and 8% ethylene glycol was improved the sperm ascrosome integrity after thawing. However, they were not significantly difference between 8% glycerol and 8% ethylene glycol on post-thawed sperm viability. Therefore, 8% ethylene glycol was more effective sperm cryoprotectant than 8% glycerol in Jeju Horse.

• Korean Journal of Ichthyology
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• v.18 no.4
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• pp.311-318
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• 2006

Ultrastructure of germ cells, the cyst epithelial cells and interstitial cells during spermatogenesis of the stone flounder, Kareius bicoloratus (Pleuronectidae) sampled on the west coast of Korea were investigated by electron microscopic observations. In the primary spermatocyte, the synaptonemal complexes appear in the zygotene stage of the prophase during maturation division. In the growing testis, especially, the interstitial cells (Leydig cells) appear near the primary, secondary spermatocytes and spermatids. Well-developed interstitial cells (steroid hormone secreting cells) which are located in the interlobular space in growing testis have three morphological characteristics of a vesicular nucleus, mitochondria with tubular cristae and smooth endoplasmic reticulum. During spermatogenesis, the primary and secondary spermatocytes attach to the cyst epithelial cell (Sertoli cell) having an elongated ovoid or triangular nucleus and several mitochondria in the cytoplasm. In the growing testis, lipid droplets, the mitochondrial rosettes and glycogen particles appear in the cytoplasm of the cyst epithelial cells near the secondary spermatocytes and spermatids. Particularly, the mitochondria, endoplasmic reticulum, little lipid droplets and the large amount of glycogen particles are present in the cytoplasm of the cyst epithelial cell in the late growing testis. In the late stage of spermiogenesis, the proximal centriole is joined to the nuclear envelope, the distal centriole forms the basal body of the flagellum and gives rise to the axial filament of the flagellum. No acrosome of the sperm is formed as seen in other teleost fish. The head of the spermatozoon is approximately $3{\mu}m$ in length and its tail is about $30{\mu}m$ in length. The axoneme of the tail flagellum of the spermatozoon consists of nine outer doublet microtubules at the periphery and two centrial singlet microtubules at the center. The spermatozoon of this species has two axonemal lateral fins. Especially, the cyst epithelial cells which located near groups of gametes in the various stages, show three functions: nutrition, phagocytosis and steroidogenesis. Especially, the nuclei of cyst epithelial cells in the recovery stage of the testicular developmental stages appear to be irregular in shape after spermiation. Of three functions of the cyst epithelial cell, several characteristics of phagocytosis are showed in the cytoplasm of the cyst epithelial cells in the recovery stage of the testicular developmental stages. At this stage, therefore, it is assumed that the cyst epithelial cells are involved in degeneration and resorption of undischarged germ cells after spermiation.

• Journal of the Korea Academia-Industrial cooperation Society
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• v.19 no.9
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• pp.116-123
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• 2018

The sperm quality is determined by the kinetic characteristics and acrosome integrity of the sperm. In the previous studies, analysis of semen quality had large errors because those experiments by using microscope had been conducted by people. In recent years, the molecular biological methods have been newly developed to complement the previous techniques. The ZAR1 gene is known to be a gene that affects early embryonic development in vertebrates, but there is no study of the association with semen. In this study, we analyzed the association between the kinetic characteristics and ZAR1 single nucleotide polymorphism (SNP) genotype. To detect the SNPs, we performed sequencing using genomic DNA from the whole bloods of Duroc pigs. We identified an SNP in the ZAR1 gene g.2540T>C. ZAR1 SNP genotypeing in 105 pigs revealed that the major and minor alleles were T and C, respectively. After we analyzed the association between the kinetic characteristics of sperm and the ZAR1 SNP genotype, we found a significant association in MOT (p<0.01), VSL (p<0.05) of the kinetic characteristics in the Duroc boars. It was confirmed that the boars with T allele were lower in MOT and VSL than C allele. Therefore, pigs with C allele are judged to be better at the MOT and VSL of semen. Based on these results, ZAR1 SNP genotyping may be a useful molecular biomarker to improve semen quality by applying molecular breeding technology.

• Korean Journal of Animal Reproduction
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• v.26 no.1
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• pp.9-16
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• 2002

This study was carried out to investigate the effects of extenders such as Beltsville thawing solution(BTS), Modena and Androhep, preservation temperature and period of liquid boar semen on semen characteristics and reproductive performance. Boars were raised at Swine Artificial Insemination Center in National Livestock Research Institute, Sunghwan, Chungnam, Korea. This experiment was carried out from 1995 to 2000. The results obtained were summarized as follows. 1. Sperm motility in the samples with Androhep and BTS reduced from day 5 and in the samples with Modena reduced from day 3 of storage. pH or 3 extenders varied from 6.24 to 7.04 during day 1 to 5 of storage. Farrowing rate of sows inseminated with liquid boar semen offended with BTs, Modena and Androhep extenders did not show any differences until day f after semen collection. Sows inseminated with Androhep extender had better farrowing rates (P＜0.05) than those with Modena extender at day 1 or 5 after semen collection, but farrowing rates after AI using BTS did not differ compared to those Androhep and Modena. Litter size did not show any differences among the three extenders, but Androhep had the decreased litter size from day i of storage. 2. Motility and normal acrosome of the sperm preserved at 5$^{\circ}C$ did not show any differences until day 4 of storage, but those at 17$^{\circ}C$ changed from day 3 and 4, respectively. Farrowing rate of sows artificially inseminated with liquid boa. semen preserved at 17$^{\circ}C$ had higher, than at 5$^{\circ}C$ (p＜0.05), but there was no significant differences in litter size. Farrowing rates and litter size were decreased from day 2 and day 3 of storage at 17$^{\circ}C$, respectively. Farrowing rate of sows inseminated with the preserved semen at 5$^{\circ}C$ did not changed until day 4, but the litter size at 5$^{\circ}C$ was lower than that at 17$^{\circ}C$.

• Korean Journal of Animal Reproduction
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• v.27 no.2
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• pp.153-161
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• 2003

This study has evaluated effect of the spermatozoa incubation on the glycosidase activity and fertilizing ability in vitro in the pig. To identify sperm glycosidases specific for sugar residues found in the zona pellucida of pig oocytes, the spermatozoa were treated experimentally and assayed for activities of $\alpha$-L-fucosidase, $\alpha$-D-mannosidase, $\beta$-D-galactosidase and N-acetyl-$\beta$-D-glucosaminidase ($\beta$-GlcNAc'ase). The glycosidases activity were higher in spermatozoa incubated for 2h than without incubation. The $\beta$-GlcNAc'ase activity was at least two-fold higher than other glycosidase regardless of spermatozoa incubation. In the same glycosidases, the activity had a tendency to increase as time of spermatozoa incubation was prolonged, but there were no differences in spermatozoa incubated during the various periods (4~24h). The percentages of spermatozoa that reached acrosome reaction were affected by glycosidases in the medium (P<0.05, for mannosidase), and were higher in spermatozoa with that than without incubation. On the other hand, the spermatozoa motility were decreased with incubation periods, but no effects by different glycosidases on the change of sperm motility during the various periods of incubation. In other experiment, the binding and penetration of pig spermatozoa were tested with oocytes matured in vitro in the presence of various glycosidase. The penetration rates were decreased with incubation of spermatozoa when oocytes were inseminated in medium with different glycosidases. These rates were higher in spermatozoa non-incubated than with incubation for 2h (P<0.05 for GlcNAc'ase; P<0.01 for control group). The sperm-zona binding rate in control group were higherthan in medium with glycosidases. In addition, the highest binding rate were obtained in medium with GlcNAc'ase. In all glycosidases, the sperm-zona binding rate in spermatozoa without incubation were higher than incubation for 2h. The significant differences were obtained in spermatozoa treated with $\alpha$-D-mannosidase (P<0.05). These results suggest that $\beta$-GlcNAc'ase is present mainly in the plasma membrane of pig spermatozoa. It was also shown that the glycosidase activity were increased in all glycosidases in spite of low sperm-zona binding rate and penetration rates by spermatozoa incubation.

• Journal of Embryo Transfer
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• v.21 no.3
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• pp.263-271
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• 2006

The purpose of this study was undertaken to compare ability of frozen-thawed sperm characteristics between two strains (miniature pig and Duroc). The semen was collected by gloved-hand method into a pre-warmed ($37^{\circ}C$) thermos bottle. The semen was diluted with same volume extender and added to LEY solution for freezing. The diluted semen was placed in 0.5 ml straws, and freezing was initiated by exposing the straws to liquid nitrogen ($LN_2$) vapours for 10 min before placing them into $LN_2$ for cryopreservation. The frozen-semen straw were thawed at 20, 37 and $50^{\circ}C$ for 1 min, 45 sec and 10 sec within water-bath. The semen sample were evaluated at 0, 3, 6, 9, and 12 h after incubation at $37^{\circ}C$ for analysis of sperm ability. Abnormality of spermatozoa in miniature pig was significantly (p<0.05) higher than that in Duroc at 0, 9 and 12 h of post-thawing incubation after frozen-thawing. The percentage of F-patterned spermatozoa in miniature pig was significantly (p<0.05) lower, while the percentage of AR (acrosome reacted spermatozoa) pattern was higher in the miniature than in the Duroc. On the other hand, there was no significant difference in the viability of spermatozoa thawed at different temperature ($20^{\circ}C\;and\;37^{\circ}C$) between two species, but the viability in miniature pig was higher (p<0.05) than in Duroc when sperm was thawed at $50^{\circ}C$. In conclusion, this study suggest that suitable freezing method for miniature pig semen is required for increasing post-thawing viability and fertilization capacity.