• Korean Journal of Animal Reproduction
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• v.14 no.4
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• pp.297-302
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• 1990

This study was carried out to investigate the effect of heparin, CSA and PC12 on sperm motility and acrosome reaction in bovine fresh and frozen semen which were washed and incubated in mTALP, and also the effect of heparin-pretreatment on motility and acrosome reaction in mTALP, and also the effect of heparin-pretreatment on motility and acrosome reaction of sperm treated with PC12, and the results obtained were as follows : 1. When fresh sperm was once washed and then incubated for 15 minutes in mTALP containing heparin 1, heparin 2, CSA and PC12, the percent of motile sperm of PC12 was significantly lower than that of control, heparin 1, heparin 2 and CSA. But the percent of acrosomereacted sperm of PC12 was signifciantly higher than that of control, heparin 1, heparin 2, and CSA. 2. When frozen sperm was once washed and then incubated for 15 minutes in mTALP containing heparin 1, heparin 2, CSA and PS12, there was no significant difference in the percent of motile sperm among treatments, but the percent of acrosome-reacted sperm of PC12 was signifciantly higher than that of heparin 2, and there was no significant difference in the percent of acrosome-reacted sperm among control, heparin and CSA. 3. When fresh sperm was twice washed and then incubated for 15 minutes in mTALP containing heparin and PC12, there was no significant differrence in the percent of motile sperm among treatments, but the percent of acrosome-reacted sperm of PC12 was significantly higher than that of control and heparin. When the sperm was incubated for 120 minutes, the percent of motile sperm of PC12 was significantly lower than that of control and heparin, but the percent of acrosome-reacted sperm of PC12 was significantly higher than that of control and heparin. 4. When fresh sperm was twice washed and preincubated in mTALP containing heparin for 0, 15, 120, and 240 minutes, and then incubated with PC12 for 15 minutes, there was no significant difference in the perce수 of motile sperm among treatments, but the percent of acrosome-reacted sperm of 120 and 240 minutes was significantly higher than that of 0 and 15 minutes.

• Korean Journal of Animal Reproduction
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• v.5 no.2
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• pp.35-42
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• 1981

A total 2,488 ejaculates during 4 years from 80 Korean native bulls over the age from 3 to 12 years of bull herds of Artificial Breeding Center, National Livestock Federatives Cooperation were collected and analyzed to study the effects of collection year, age of bulls, month of years, collection interval and ejaculation frequency per day on semen volume, sperm concentration, total sperm per ejaculate and sperm motility. 1. Semen volume, sperm concentration, total sperm per ejaculate and sperm motility index for the all ejaculates using least squares procedure averaged 5.73ml, 9.133${\times}$108/ml, 52.527${\times}$108 and 62.81, respectively. 2. Semen volume varied significantly with collection year, collection interval and ejaculation frequency per day(P<0.01), but effects of age of bulls and month of years on semen volume were not significant. Eight to 15 days collection interval showed the highest volume, and 1st ejaculate contained 5.6% more volume than 2nd ejaculate. 3. There were significant differences among collection years, months of years and ejeculation frequencies per day except collection intervals in sperm concentration per ml(P<0.05, P<0.01). Six to 8-year-old bulls was the highest concentration. Higher sperm concentration per ml was in April to July and lower month was October and December. Sperm concentration in 2nd ejaculate was higher than in 1st ejaculate. 4. Total sperm per ejaculate affected by all environmental factors studied(P<0.05, P<0.01). Age of bulls, collection interval and ejaculation frequency per day showed the highest total sperm was 6 to 8-year-old bulls, 8 to 15 days interval and 1st ejaculate, respectively. Higher total sperm per ejaculate was in April to July and lower total sperm was in September to December. 5. In sperm motility, there were significant differences among collection years, ages of bulls and collection intervals except months of years and ejaculation frequencies (P<0.01). Higher sperm motility was in 6 to 12-year-old bulls and in 5 to 7 days collection interval.

• Proceedings of the KSAR Conference
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• 2001.03a
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• pp.56-56
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• 2001

The exogenous gene transfer by intracytoplasmic sperm injection (ICSI) procedure has been recently used to produce transgenic mice and pigs. Sperm-mediated DNA transfer has the potential to markedly simplify the generation of transgenic animals. This method may serve as an alternative to the pronucleus injection of DNA for the production of transgenic pigs. Therefore, in this study, we investigated the expression of transgene after co-injection of spermatozoon or sperm head with green fluorescent protein (GFP) gene into in vitro matured porcine oocytes. Spermatozoon and sperm head, that was obtained by sonication, were treated with 0.03% Triton X-100 to remove the membrane. They were preincubated with linearized pEGFP-N1 for 1 min, and then embryos cultured NCSU23 medium for 2.5 days after co-injected of sperm and DNA. We monitored expression of GFP in embryos under epifluorescent microscope. The remove of sperm membrane did not alter the developmental competence of embryos after ICSI. At 7 days following injection, the rates of blastocysts following injection of intact sperm (15.0%), and of sperm with disrupted membrane (14.2%) were higher than that following IVF (10.0%). Porcine oocytes injected with sperm which co-cultured with DNA concentration of 1, 0.1, and 0.01 ng were 60, 65.7 and 75% and 18.5, 37.4 and 22.2% for rates of cleavage and GFP expression, respectively. In vitro matured porcine oocytes injected with sperm and isolated sperm head resulted in 69 and 59.7% of cleavage rates, respectively The rates of embryo GFP expressed did not significantly different between sperm (20.4%) and sperm head (20.0%) injection. The transgenic embryos with the clusters of positive blastomeres were observed under fluorescent microscope. Most of embryos expressed GFP gene showed mosaicism. They showed GFP expression at 1/4, 2/4 and 3/4 of blastomeres at the 4-cell stage. Among these 4-cell embryos, the expression rate of 1/4 blastomere group (54.6%) was higher than the other groups (15.3-30.7%). These results indicate that membrane disrupted sperm could attach with exogenous DNA, and that this procedure may be useful to introduce foreign gene into porcine oocytes. Therefore, our data suggest that the ICSI car be a useful tool to efficiently produce transgenic pig as well as other mammals.

• Proceedings of the KSAR Conference
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• 2003.06a
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• pp.26-26
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• 2003

Hyaluronic acid (HA)-binding sites have been shown the diagnostic potential fur assessment of sperm maturity, which is related to male fertility. This study was designed to evaluate chromosomal patterns in porcine embryos produced by in vitro fertilization (IVF) and intracytoplasmic sperm injection (ICSI) with non- or HA-binding sperm (HABS). For binding of sperm with HA, sperm incubated in 10 ${mu}ell$ drop containing HA (0.8 mg/ml)-agarose (0.8%) mixture for 15 min. IVF and ICSI with non- or HA-bound sperm examined with matured oocytes at 44 hr after in vitro maturation. Embryos were cultured in 50 ${mu}ell$ of NCSU 23 containing 0.5% BSA for 5 days and then in 50 ${mu}ell$ of NCSU 23 containing 10% FBS for 2 days. For the evaluation of chromosomal aneuploidies, chromosome 1 sub-metacentric specific probe was used in sperm and embryos by fluorescence in situ hybridization (FISH). The frequency of aneuploidy sperm for chromosome 1 was 6.25%. The significant differences following IVF and ICSI with non- or HA-bound sperm were not observed in blastocyst formation rates (18.6, 23.5, and 23.8%) and cell number (61.8 $\pm$ 12.5, 55.5 $\pm$ 7.3, and 59.3 $\pm$ 9.6). Moreover, the percentage of diploidy in 4-cell stage embryos was 57.1% (IVF), 68.8% (ICSI), and 76.3% (ICSI-HABS). These results suggest that HA-binding sites may be a material for selection of normal sperm for ICSI. Therefore HA selection of normal sperm may be reduce the loss to embryonic mortality prior to embryo transfer in pig.

• Clinical and Experimental Reproductive Medicine
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• v.42 no.3
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• pp.101-105
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• 2015

Objective: Several publications have established a relationship between sperm DNA damage and male factor infertility, based on data from America, Europe, and Asia. This study aimed to compare the extent of sperm DNA damage in sperm samples from Nigerian men with unexplained infertility and in sperm samples from a fertile group composed of sperm donors who had successfully impregnated a female partner naturally or through assisted conception. Methods: A total of 404 men underwent male fertility evaluation at Androcare Laboratories and Cryobank participated in this study. Semen analysis and a sperm chromatin structure assay (SCSA) were performed on all subjects. Results: The men in the unexplained infertility group were slightly older than the men in the fertile sperm group ($36{\pm}10$ years vs. $32{\pm}6$ years, p=0.051). No significant difference was observed between the two groups in semen analysis parameters ($p{\geq}0.05$). Men in the unexplained infertility group with normal semen parameters had a significantly higher DNA fragmentation index (DFI) than men in the fertile sperm group ($27.5%{\pm}7.0%$ vs. $14.1%{\pm}5.3%$, p<0.05). In the unexplained infertility group, 63% of the men had a DFI greater than 20%, compared to 4% in the fertile sperm group. In the unexplained infertility group, 15.2% of the subjects had a DFI greater than 30%, compared to 1% in the fertile sperm group. Conclusion: Our study showed that the SCSA may be a more reliable predictor of fertility potential than traditional semen analysis in cases of unexplained infertility.

• Development and Reproduction
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• v.7 no.2
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• pp.81-88
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• 2003

Initiation and activation of sperm motility are prerequisite processes for the contact and fusion of male and female gametes at fertilization. The phenomena are under the regulation of CAMP and $Ca^{2+}$ in vertebrates and invertebrates. Mammalian sperm requires $Ca^{2+}$and cyclic AMP for the activation of sperm motility. Cell signaling for the initiation and activation of sperm motility has been well studied in the ascidians, Ciona intestinalis and C. savignyi and salmonid fishes. In Ciona, whose cell signaling for activation of sperm motility has been established, the sperm-activating and -attracting factor released from unfertilized egg requires extracellular $Ca^{2+}$ for activating sperm motility and eliciting chemotactic behavior of the activated sperm toward the egg. On the other hand, the cyclic AMP-dependent phosphorylation of protein is essential for the initiation of sperm motility in salmonid fishes. A decrease in the environmental Ti concentration surrounding the spawned sperm causes a li efflux and $Ca^{2+}$ influx through the specific $K^{+}$ channel and dihydropyridine-sensitive L-/T- type $Ca^{2+}$ channel, respectively, thereby leading to the membrane hyperpolarization and $Ca^{2+}$ influx. The membrane hyperpolarization synthesizes cyclic AMP, which triggers the luther Process of cell signaling, i.e., cyclic AMP-dependent protein phosphorylation, to initiate sperm motility in salmond fishes.almond fishes.

• Journal of Embryo Transfer
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• v.15 no.1
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• pp.85-94
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• 2000

These experiments were conducted to examine the effects of theophylline, pentoxifylline and heparin on frozen-thawed Hanwoo sperm for enhancing motility and viability of sperm. Frozen-thawed semen collected from one bull was treated in TALP(tyrode-albuminlactate-pyruvate) containing varous concentrations of theophylline and pentoxifylline. After incubated at 5% CO2 in air atmosphere for 6 hours, the motility of sperm after the treatments was characterized by CASA(computer aided semen analysis) system. When monitored notility(MOT) and curvilinear velocity(VCL), theophylline and pentoxifylline exerted their optimal action at the concentration of 30 mM and 3 mM, respectively. No difference of sperm motility was observed when the sperm was treated with both substances compared with a single treatment of each substance. Comparison was then made for evaluating the effect of theophylline and / or pentoxiophylline on the motility and viability of significant treatment effects of each substance, high MOT and VCL values were detected in sperm treated with theophylline. In the case of sperm viability examined by an eosin-nigrosin staining, however, a significant decrease was found after the combined treatment of theophylline+pentoxyphilline than after the treatment with heparin alone or no treatment(P<0.05). In conclusion, theophylline, pentoxiphylline or heparin can be used for enhancing the motional characteristics and viability of frozen thawed Hanwoo semen. Considering characteristics of these substances, theophyline may be useful in the artificial insemination system, which requires vigorous sperm motility. While, heparin supporting sperm viability in vitro can be effectively used for improving in vitro-fertilization system.

• Korean Journal of Veterinary Research
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• v.39 no.1
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• pp.220-225
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• 1999

The aim of these experiments was to investigate the effects of bovine oviduct epithelial cells (OEC) derived from different segments to bind sperm binding and maintain their motility in vitro. In experiment 1, the number of sperm attached to OEC derived from isthmus or ampulla, the motility of unattached sperm during co-culture and fertilizing ability were assessed. In experiment 2, heparin treated sperm (hsp) or no treated sperm (nsp) were used to evaluate OEC binding ability of capacitated sperm. In experiment 1, regardless of their origin, approximately 65% of the sperm were attached to OEC within 2h. From 6h of co-culture, the numbers of unattached sperm on ampullary OEC were significantly higher than those on isthmic OEC (p<0.005). From 12h of co-culture, the motility of unattached sperm on isthmic OEC were significantly higher than those on ampullary OEC(p<0.05). The cleavage rate of oocytes inseminated on OEC derived from isthmic segment was also significantly higher than those from ampullary segment (p<0.01). In experiment 2, the numbers of unattached hsp on OEC were significantly higher than those of controls (p<0.01), between 2-24h examination. From 12h of co-culture, the motility of unattached nsp were significantly greater than those of hsp (p<0.01). These results show that bovine OEC derived from the isthmus play more important role(s) for sperm binding, maintaining motility and fertilization in vitro than those from the ampulla, and heparin induced capacitation may change sperm binding ability on OEC in vitro.

• Applied Microscopy
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• v.30 no.1
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• pp.21-44
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• 2000

This study was carried out to investigate sperm storage, and the fate of spermatozoa in the female reproductive tract during hibernation in Korean greater horseshoe bat, Rhinolophus ferrumequinum korai. (1) Numerous sperm occurring in uterine lumen and glands were engulfed, and disappeared by the polymorphouclear leucocytes during the hibernation. (2) The stored sperm present in caudal isthmus of oviduct only, the heads of sperm toward the oviductal epithelial cells. Therefore, the projected sperm during the mating season are only alive in the caudal isthmus of oviduct in the long hibernation. The present result suggests that the caudal isthmus of oviduct may play an important role as the principal storage site in capacitation of sperm. (3) In March, the sperm do not occur in the caudal isthmus of oviduct. It suggests that the stored sperm in the caudal isthmus of oviduct should migrate to the ampulla of the site of fertilization to meet ovum in the period of ovulation. The results of this experiment consider that prolonged sperm storage, fate of sperm and sperm migration in the long hibernation have a kind of mechanism for the fertilization.

• Applied Microscopy
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• v.28 no.2
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• pp.193-205
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• 1998

The Urechis unicinctus sperm and spermatogenic cells prepared from the testis are investigated to identify $\alpha-tubulin$ of axoneme microtubules using mouse monoclonal $anti-\alpha-tubulin$ as the first Ab and Gold(10nm) conjugated goat anti-mouse IgG as the Ab marker. The Ag-Ab reaction analyzed excellently the localization of $\alpha-tubulin$ and the gold particles incorporated with the proximal and distal centrioles, manchette microtubules, and flagellum. The gold particles can be also observed in the spermatogenic cells while the cells are still in sperm ball which is composed of a somatic cell and spermatogenic cells. The sperm ball is the functional unit of sperm production in U unicinctus testis. The spermatids are developed from the spermatogenic cells in the sperm ball and released into the testis cavity through a cortical cytoplasmic opening. The spermatid architectures are similar with the mature sperm of the testis cavity in aspects of shape of discoid acrosome, degree of nuclear condensation and ring type of mitochondrion. However, the distal centriole connecting with the flagella can be observed from the mature sperm while the both proximal and distal centrioles reveal only in the spermatids. The proximal centriole is directly connected with nuclear outer membrane during the stage of nuclear condensation and oriented perpendicularly to the distal centriole whose axis coinciding with the longitudinal axis of the spermatozoon. There are indications that the distal centriole is intimately associated with the polymerization of the flagellum. The manchette microtubules appear during spermatid development but the mature sperm have round head and no conspicuous middle piece.