• Korean Journal of Animal Reproduction
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• v.15 no.3
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• pp.195-199
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• 1991

This study was carried out ot investigate effect of pH stimulation on acrosome reaction of bovine spermatozoa. The results obtained were as follows : 1. When sperm was sequentially washed with SHP solution of pH 7.4, 7.7 and 7.4 and incubated in mTALP solution of pH 7.4 for 120min, 15, 30, 60 and 120min incubations showed significantly(p<0.05) higher sperm acrosome reaction rate than 0 min. 2. When sperm was sequentially washed with SHP solution of pH 7.4, 8.0 and 7.4 and incubated in mTALP solution of pH 7.4 for 15 minutes, sperm acrosome reaction rate was significantly(p<0.01) increased until 9 min. Incubation, but not increased thereafter. 3. When sperm were separately washed with SHP solutions of pH 7.0, 7.4 and 8.0 and incubated in mTALP solution of pH 7.4 for 9min, sperm acrosome reaction rate was 74.8, 71.8 and 93.4%. pH 8.0 showed signifciantly(p<0.01) higher sperm acrosome reaction rate than pH 7.0 and 7.4. The results suggest that stimulation of sperm with high pH induces sperm crosome reaction.

• Korean Journal of Animal Reproduction
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• v.10 no.2
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• pp.182-187
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• 1986

As a fundamental study to increase the fertility and to modify the sex ratio in cattle, highly motile sperm were separated by bovine serum albumin gradients. The pregnancy rates of Korean native cow and Holstein cow, and the sex ratio between AI and natural mating were also investigated. The results obtained were as follows. 1. First service pregnancy rate of Korean native cow in artificial insemination was higher than that of Holstein. 2. At secondary sex ratio in artificial insemination and natural mating, male ratio in artifical insemination was slightly higher than that in natural mating. 3. The sperm separated from marketed frozen semen using 6%, 10% and 20% bovine serum albumin showed significantly high value in motility, percent of normal sperm and progressive motility as compared with control sperm.

• Journal of Embryo Transfer
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• v.12 no.2
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• pp.161-169
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• 1997

This study was conducted to compare the insemination time of bovine oocytes and determine the effects of glucose(1.5 mM) on the development of bovine embryos at early cleavage stage. Oocytes were matured for 24 h, followed by exposure to sperm and cultured in modified Tyrode's media drops or with bovine oviduct epithelial cell monolayer prepared in TCM199(BOECM). Insemination time and culture system were varied in each experiment. In experiment 1, to investigate the developmental capacity of bovine embryos after different time of exposure to sperm, bovine ova and sperm were co-incubated for 18, 30 or 54 h, respectively. The development to blastocysts of 30 and 54 h insemination groups were significantly higher(P<0.05) than 18 h group, and in case of blastocysts of cleaved embryos, 30 h group were significantly higher(P<0.05) than other groups. In experiment 2, we investigated the effect of glucose on early bovine embryos. After 18 h insemination, in vitro fertilized oocytes were separated following 3 groups ; G+0, C+24 and C+48. Oocytes of G+0 group were cultured in glucose added Tyrode's medium after fertilization, oocytes in C+24 and C+48 groups were cultured in glucose free Tyrode's medium after fertilization. After 24 h culture, G+24 group was moved to glucose added medium. All oocytes of 3 groups were moved to BOECM after 48 h culture. The rates of cleavage and development to blastocysts in G+0 group were significantly lower than other groups. In experiment 3, we determined the effects of glucose exposure from 8 to 20 h after insemination on the cleavage and development of oocytes. The oocytes in glucose added group had high capacity of cleavage and further development. This study shows that in bovine oocytes, the optimal exposure to sperm is 30 h and glucose exposure to bovine one-cell embryos is detrimental to their first cleavage and further development in vitro but there has no evidence of detrimental effect of glucose(1.5 mM) exposure to bovine embryos over the two-cell stage in vitro.

• Journal of Embryo Transfer
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• v.12 no.2
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• pp.219-226
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• 1997

Follicular fluid influxed into the oviduct during ovulation may affect movement of sperm for fertilization Thus, in this study, the effect of follicular fluid, obtained from follicles of l0mm in diameter, on number and quality of sperm recovered by swim-up separation was investigated and sperm-movement stimulating components extracted from follicular fluid with methanol and isooctane were separated by gel filtration with Sepadex G-1O, G-25 and G-1OO gels, and were isolated by electrophoresis with SDS-PAGE mini gel. The results obtained were as follows; 1. Diluted follicular fluid stimulated sperm movement. 2. Sperm-movement stimulating factors were in methanol extract. 3. Sperm-movement stimulating effect of methanol extract appeared in fraction I among fractions recovered after gel filtration. And the fraction I contained proteins indicating 4 major bands as about 47, 43, 25 and 14 kilodaldons and 5 minor bands as about 67, 58, 23, 22 and 21 kilodaldons. 4. The fraction I recovered from G-100 gel showed significantly low percentage of motile sperm and had no protein indicating the band of 67 kilodaldons among the minor bands.

• Journal of Veterinary Clinics
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• v.7 no.2
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• pp.497-500
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• 1990

This study was carried out determine whether Unopette can be used for the observation of sperm morphology and sperm Concentration. Rabbit sperm and frozen-thawed bovine sperm were observed with phase contrast microscope after dilution with Unopette acooriding to duration of preservation at 3～5$^{\circ}C$. Sperm using Unopette showed high normal sperm(％) than sperm using hematoxylin-eosin until 48 hours. Sperm using Unopette revealed no difference in sperm concentration until 24 hours, as compared with control sperm. As a result, Unopette was assessed as appropriate solution for preservation in terms of morphological observation and sperm concentration.

• Journal of Animal Science and Technology
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• v.65 no.6
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• pp.1205-1213
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• 2023

The goals of the present study were to develop a simple method for obtain highly purified pig sperm hyaluronidase (pHyase) and to assess its activity, function, and safety. In mammals, sperm-specific glycophosphatidylinositol (GPI)-anchored Hyase assists sperm penetration through the cumulus mass surrounding the egg and aids in the dispersal of the cumulus-oocyte complex. Recently, Purified bovine sperm hyaluronidase (bHyase) has been shown to enhance therapeutic drug transport by breaking down the hyaluronan barrier to the lymphatic and capillary vessels, thereby facilitating tissue absorption. Commercially available Hyase is typically isolated from bovine or ovine; which have several disadvantages, including the risk of bovine spongiform encephalopathy, low homology with human Hyase, and the requirement for relatively complex isolation procedures. This study successfully isolated highly purified pHyase in only two steps, using ammonium sulfate precipitation and fast protein liquid chromatography. The isolated Hyase had activity equal to that of commercial bHyase, facilitated in vitro fertilization, and effectively dissolved high molecule hyaluronic acid. This simple, effective isolation method could improve the availability of pHyase for research and clinical applications.

• Journal of Embryo Transfer
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• v.7 no.1
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• pp.21-30
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• 1992

This study was aimed to develope the new method for bovine sperm capacitation through testing and combinating factors relating to sperm capacitation. For verifying the efficiency of this method on inducing capacitation, in vitro fertilization was carried out. The results obtained were as follows: When pHs of HBS /PR for sperm-washing were 5.99, 6.38, 6.78, 7.10, 7.40, 7.69, 8.15, 8.45 and 8.83, variances between light absorhance differences obtained from sperm pre-washing solution and post-washing solution(VADs) were 0.000, 0.001, -0.001, -0.005, -0.005, -0.021,-0.017,-0.016,and-0.036, respectively. There were significant decreases of VADs in pH 7.69 and pH 8.83. When sperm were firstly, secondly, and thirdly washed with HBS /PR, VADs were -0. 024, - 0.006, and - 0.004, respectively. There was significant decrease of VAD in 1st sperm-washing. When washed-sperm were secondly washed with HBS /PR supplemented with no (con-trol), heparin, CSA, PC12, and BSA, VADs were 0.009, -0.024, -0.008, -0.009, and 0.014, respectively. When the sperm were thirdly washed with HBS /PR with no(control), heparin, CSA, PC12 and BSA, VADs were 0.020, -0.007, 0.005, 0.006, and 0.019, respectively. Only heparin treatment showed the negative values of VADs in 2nd and 3rd sperm-washing. Of oocytes cultured with sperm which were repeatedly washed with heparin and high pH, 52% (57/110) were cleaved over 2 cell stage. However, percent of oocytes parthenogenetically divided was 5%(2 /42).

• Journal of Embryo Transfer
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• v.31 no.3
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• pp.299-305
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• 2016

Although cryopreservation of sperm is routinely used for clinical requirement, it has some problems, such as high generation of reactive oxygen species (ROS) and cold-shock. To reduce the detrimental damage in sperm, anti-oxidants were added to cryoprotectant for sperm. Curcumin is one of anti-oxidants, which are added in cryoprotectants. However, recent studies have demonstrated that curcumin decreases sperm viability and motility. This study was performed to identify the effect of curcumin on hydrogen peroxide ($H_2O_2$)-exposed bovine sperm, which were cryopreserved-thawed. In $H_2O_2$-exposed bovine sperm, reactive oxygen species (ROS) were significantly reduced by treatment with curcumin in a dose-dependent manner (p < 0.05). Among tested concentrations of curcumin (1 to $50{\mu}M$), 30 and $50{\mu}M$ curcumin showed anti-oxidant effect on $H_2O_2$-induced ROS generation. On the other hand, combination of 30 or $50{\mu}M$ curcumin with anti-oxidant $H_2O_2$ increased the percentage of apoptotic sperm compared to only $H_2O_2$ treatment. Sperm viability was also decreased in the combination of 30 or $50{\mu}M$ curcumin with $H_2O_2$ as judged by FDA/PI staining. $H_2O_2$-induced decrease in sperm progressive motility was recovered by treatment with $1{\mu}M$ curcumin. These results show that high concentration of curcumin has anti-oxidant effect, but it has also cytotoxic effect on bovine sperm. Sperm viability and motility might be more affected by cytotoxic signals of curcumin compared to antioxidant signals.

• Clinical and Experimental Reproductive Medicine
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• v.24 no.3
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• pp.301-309
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• 1997

The objective of this study was to determine the microtubule assembly and chromatin configuration during the first cell cycle in bovine oocytes following injection of spermatozoon, sperm head and tail. The microtubule and chromatin configuration was imaged with fluorescent labeled monoclonal ${\alpha}$-tubulin antibody and propidium iodide under laser scanning confocal microscope. Microtubule and chromatin dynamics in bovine oocytes following intracytoplasmic sperm injection (ICSI) were not different from those observed during in vitro fertilization (IVF). Following ICSI, the microtubular aster was observed around sperm midpiece. During pronuclear formation, the sperm aster was enlarged and seen around male and female pronuclei. At mitotic metaphase, the microtubular spindle assemble astral poles and chromosomes were aligned on the spindle equator. At mitosis, asters were concentrated to each spindle pole and they filled the cytoplasm. After injection of the isolated sperm head, the microtubular aster was not seen around sperm head in any cases (0/18). Instead, microtubules were organized from the cytoplasm, which filled the whole cytoplasm during pronuclear apposition. These microtubules seem to move male and female pronuclei. These results suggest that isolated sperm head can develop into normal pronucleus in mature bovine oocytes, and competent to participate syngamy with the ootid chromatin. The functional microtubules following isolated sperm head injection in bovine oocytes appeared to be organized solely from maternal stores.

• Korean Journal of Animal Reproduction
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• v.25 no.4
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• pp.409-419
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• 2001

This study was to examine whether the in vitro friability, motility and intact acrosome of frozen-thawed bovine and human sperm can be improved by adding Pentoxifylline (PF) or Fertilization Promoting Peptide (FPP). Human semen was frozen ultra-rapidly using Test yolk-buffer (TYB) freezing medium. Additive (PF, FPP) effects in frozen-thawed bovine and human sperm were analyzed by microscopic count for sperm motility and coomassie brilliant blue staining method f3r sperm acrosome intact. The in vitro motility of frozen-thawed bovine sperm with 5 mM PF treatment group (50.0%) was significantly higher than that of control (34.0%) (P＜0.05). In the frozen-thawed bovine sperm was examined, the intact acrosome rate of 50 nM FPP treatment (49.0%) was significantly higher than those of control (30.0%) and 25 nM FPP (38.0%) treatment groups (P＜0.01). In human semen, when in vitro motility of sperm with PF addition prior to freezing was examined, the result of 5 mM treatment group (51.0%) was significantly higher than those of control and 2.5 mM treatment group (39.0, 40.0%) (P＜0.01). In addition, 50 nM (75.5%) FPP adding in all treatment procedures for human semen freezing (before freezing, freezing and after thawing) was significant effect on maintenance of the sperm intact acrosome percentage (control: 45.0; 25 nM: 53.0; 100 nM: 68.0%) (P＜0.01). Also, the intact acrosome rate of human sperm with FPP (65.0%) was significantly higher than that with PF (43.0%) (P＜0.05), although sperm motility was slightly higher in PF treatment group. These results suggest that improved sperm motility and intact acrosome of frozen thawed bovine and human sperm can be obtained by addition of PF or FPP, and that the enhanced in vitro viability, motility and intact acrosome can be obtained by addition of FPP in all semen freezing procedures.