• Korean Journal of Poultry Science
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• v.42 no.2
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• pp.109-116
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• 2015

Ogye rooster sperm chromatin status can be detected using well established sperm assays. In this paper, a simple and fast method to monitor rooster sperm chromatin status could be employed in field for assessment of chicken sperm quality. Using standard bright field microscope, Diff-Quik stains can be reproducibly, easily and routinely monitored with simple staining. The presence of abnormal chromatin staining of rooster sperm was determined by darker stain in head. In the fresh semen, the viabilities of three tested Ogye spermatozoa were 93.53%, 82.42% and 90.63% and normal chromatin rates were 87.96%, 74.25% and 85.10% respectively. However, after freezing, the rates of viability of thawed semen were reduced to 69.58%, 61.98% and 72.20% and normal chromatin rate also reduced to 58.91%, 48.49% and 63.34%. A significant correlation between live sperm and normal sperm nuclei was 0.875 in fresh semen and 0.513 in frozen semen. After incubation of sperm at $37^{\circ}C$ for 5min, the rates of viability, chromatin normality and sperm head activity were shown as $90.63{\pm}1.28%$, $82.44{\pm}8.09%$ and $66.68{\pm}10.29%$ in fresh semen. However, the rates of thawed semen were reduced to $67.92{\pm}7.55%$, $56.92{\pm}12.15%$ and 47.32{\pm}5.02%, respectively. The relationship between chromatin normality and sperm head movements in fresh and thawed semen were 0.564 and 0.540, respectively. With these results, the chicken sperm normality could be assessed by the Diff-Quik staining that could be used for chromatin status of sperm head and activated morphology of live spermatozoa, as a simple and rapid staining method.

• Journal of Embryo Transfer
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• v.30 no.1
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• pp.45-50
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• 2015

The purpose of this study is to produce wanted sex progeny of genetically confined White Hanwoo (albinism) with preselected sex sperm. One bull of White Hanwoo was chosen for semen donor and X sperm was sorted by MoFlo XDP cell sorter. To compare the pregnancy and birth rates, KPN straw was used as control, total number of unsorted sperm was $20{\times}10^6/straw$. Sexed X frozen semen with $20{\times}10^6$ cells or $4{\times}10^6$ cells per straw were in seminated twice on Hanwoo heifers. The abnormality of the sexed X semen was $24.9{\pm}7.31%$ and distal reflex abnormality of mid piece was significantly (p<0.05) higher (11.7%) compared with that of KPN 768 (5.6%). There were no differences on the pregnancy and birth rates between $2{\times}10^6$ cells or $4{\times}10^6$ cells of X-sperm but KPN semen showed significant differences (p<0.05). The pregnancy rates of KPN 768, $2{\times}10^6$ cells and $4{\times}10^6$ cells X-sperm of White Hanwoo cattle were 85.0%, 26.3% and 50%. The birth rates were 80.0%, 15.8% and 21.4%, respectively. The female offspring rates of KPN 768, $2{\times}10^6$ cells and $4{\times}10^6$ cells X-sperm of White Hanwoo cattle were 43.8%, 100% and 100% (p<0.05). These results indicated that sex sorted White Hanwoo could be used for the production of wanted progeny with $2{\times}10^6$ cells/straw for AI. To increase the efficiency of calf production, the sperm number of sex sorted semen will be optimized for sex selection of White Hanwoo progeny.

• Reproductive and Developmental Biology
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• v.35 no.3
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• pp.363-367
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• 2011

The purpose of this study was to improve of frozen-thawed sperm using magnetized water in Korean native cattle. Before cryopreservation, without egg-yolk Triladyl$^{(R)}$ solution was flowed though magnet [0, 2000, 4000 and 6000 Gauss(G)] for S min. The freezing of dilluted semen added with Triladyl containing 20% egg-yolk. Analysis of frozen-thawed sperm was estimated viability with SYBR14/PI double stain, membrane intact with hypoosmotic swelling test (HOST), acrosome reaction with FITC-PNA, mitochondria membrane function with Rhodamin 123 by flow- cytometry. Sperm viability was significantly higher in 4000G group than other groups (p<0.05). However, the Hypoosmotic Swelling Test(HOST) was significantly higher in fresh, 4000 and 6000G than 0 and 2000G (p<0.05). In addition, mitochondria membrane damage and acrosome damage were significantly lower in 6000G group than other groups (p<0.05). in conclusion we suggest that magnetized water could be improve ability of sperm on cryopreservation in Korean native cattle.

• Journal of Veterinary Clinics
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• v.35 no.2
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• pp.39-45
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• 2018

We evaluated the effects of green tea extract (GTE) supplementation at different dilution steps on boar sperm freezing and in vitro fertilization. Sperm intracellular hydrogen peroxide ($H_2O_2$), motility, viability, acrosome integrity and morphology were determined. In addition, sperm IVF parameters (penetration and monospermy) and glutathione (GSH) levels of presumptive zygotes (PZs) were evaluated. Semen was diluted in lactose egg yolk (LEY) and cooled at $5^{\circ}C$ for 3 h (first dilution step) and then diluted in LEY with 9% glycerol and maintained at $5^{\circ}C$ for 30 min (second dilution step). Four experimental groups were compared: first and second dilution steps without GTE (control), first dilution step with GTE (Step 1), second dilution step with GTE (Step 2) and first and second dilution step with GTE (Step 1+2). The spermatozoa were frozen in nitrogen vapor. Higher sperm motility, viability and acrosome integrity after thawing were observed in Step 1, Step 2 and Step 1+2 groups compared with the control (P < 0.05). Lower $H_2O_2$ level was observed in Step 1+2 compared with control and Step 1 (P < 0.05). For IVF, matured oocytes were co-cultured with spermatozoa frozen according to the experimental groups. GSH levels of PZs were significantly higher in Step 2 and Step 1+2 than in control and Step 1 (P < 0.05) without a significant difference in IVF parameters. In conclusion, supplementation with GTE in both first and second dilution steps during the freezing process resulted in better boar sperm cryopreservation and might be beneficial for further embryo development.

• Korean Journal of Animal Reproduction
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• v.10 no.1
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• pp.91-99
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• 1986

This experiment was undertaken to examine the effects of HIS treatment on the motility and acrosome reaction of frozen bovine spermatozoa and to test their abilities to interact with zona-free hamster eggs in vitro. Also, in vitro results were compared with those of bull's fertility in AI. The frozen semen from four Holstein bulls were exposed to HIS-DM for 5 minutes after thawing and then preincubated for 60 minutes in DM prior to insemination. The hamster eggs were mounted, fixed and stained 6 hours after exposure to boving spermatozoa and examined under a phase-contrast microscope. 1. The sperm motility expressed as a mobility index dro, pp.d significantly from 60-75 to 12-24 after exposure to HIS-DM, but increased in 32 to 41 at insemination. Bull C showed a low motility index than those of the orher bulls. The percentage of acrosome reaction by staining procedure were increased by HIS-DM treatment but did not change during 7 hours incubation period in DM. 2. The overall percentage of hamster eggs interacting with bull spermatozoa was 56.3%, 58.3%, 66.6% and 70.0%, respectively. Although there was no significant difference among bulls in the penetration rate of spermatozoa into hamster eggs, high proportions of eggs interacted with spermatozoa from Bull C and D than those from Bull A and B. 3. The conception rates (60-90 day RP) resulting from AI were 62.5%, 67.5% and 70.9% for Bull A, B and C, respectively. These results were in good agreement with the invitro results that the proportions of bull sperm-egg interction were greater for Bull C than for Bull A and B.

• Clinical and Experimental Reproductive Medicine
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• v.45 no.1
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• pp.44-47
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• 2018

Onco-testicular sperm extraction is used to preserve fertility in patients with bilateral testicular tumors and azoospermia. We report the case of a testicular tumor in the solitary testis of a patient who had previously undergone successful contralateral orchiectomy and whose sperm was preserved by onco-testicular sperm extraction. A 35-year-old patient presented with swelling of his right scrotum that had lasted for 1 month. His medical history included a contralateral orchiectomy during childhood. Ultrasonography revealed a mosaic echoic area in his scrotum, suggesting a testicular tumor. The lesion was palpated within the normal testicular tissue along its edge and semen analysis showed azoospermia. Radical inguinal orchiectomy and onco-testicular sperm extraction were performed simultaneously. Motile spermatozoa were extracted from normal seminiferous tubules under microscopy and were frozen. Eventual intracytoplasmic sperm injection using the frozen spermatozoa is planned. Onco-testicular sperm extraction is an important fertility preservation method in patients with bilateral testicular tumors or a history of a previous contralateral orchiectomy.

• Journal of Embryo Transfer
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• v.18 no.3
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• pp.195-201
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• 2003

This study was carried out to establish most suitable freezing condition, to evaluate the different condition of freezing and thawing rates on the viability and motility of frozen canine spermatozoa. The ejaculated semen was added to obtained 200∼400 ${\times}$ 10$^{6}$ /$m\ell$ with extender I and was cooled to 4$^{\circ}C$ over 30, 60 and 120 minutes. And then, semen was diluted with extender II containing 4, 6 and 8％(v/v) glycerol for 60 min, respectively and packaged in 0.5$m\ell$ straw, equilibrated far 30, 60 and 120 min at 4$^{\circ}C$ and cryopreserved in liquid nitrogen vapor at different distance(3, 5, 7 and 9 cm, respectively), plunged into nitrogen tank. Samples were thawed by placing straws into 27, 37, 47, 57$^{\circ}C$ water bath for 120, 20 and 12 sec, respectively. The results were as follows; 1. The survival and motility rate immediately post-thawing was significantly higher in samples frozen in 4％ glycerol than 6 or 8％ glycerol(P< 0.05). 2. According to equilibration time at 4$^{\circ}C$, the survival and motility rate immediately post-thawing was significantly higher in samples frozen after 60 min equilibration than 30 or 120 min equilibration(P<0.05). 3. Freezing in distance of 5 cm from liquid nitrogen yield better survival and motility rate than the others(3, 7 or 9 cm)(P<0.05). 4. The effect of thawing rate on sperm survival were higher when the thawing was done at 37$^{\circ}C$ for 120 sec(P<0.05).

• Clinical and Experimental Reproductive Medicine
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• v.36 no.1
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• pp.35-43
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• 2009

Objectives: Seminal concentration of tumor necrosis factor-alpha (TNF-${\alpha}$) relevant to sperm nuclear DNA integrity has not been studied. The present study aimed to evaluate seminal concentration of TNF-${\alpha}$ in correlation with sperm parameters and nuclear DNA integrity in asymptomatic healthy donors. Methods: Semen samples were obtained by masturbation from forty-five healthy donors. Results: Sperm quality was assessed by computer-assisted semen analysis and nuclear DNA integrity measured by the TUNEL assay in raw semen. TNF-${\alpha}$ concentrations were measured by ELISA in frozen-thawed seminal plasmas. Sperm DNA fragmentation rates were ranged between 1.9% and 53.0% (mean${\pm}$SD, 12.4${\pm}$9.6%). Univariate analysis revealed that DNA fragmentation rate was not associated with sperm concentration or motility but had a correlation with linearity negatively (r=-0.325, p=0.03) and age positively (r=0.484, p=0.001). The mean seminal concentration of TNF-${\alpha}$ was 4.9 pg/mL with a range from 1.1 to 22.6 pg/mL. The TNF-${\alpha}$ concentration had no correlation with clinically relevant parameters of sperm quality or nuclear DNA fragmentation rate. Conclusion: Our results indicate that sperm nuclear DNA fragmentation may be not associated with seminal TNF-${\alpha}$ level or sperm quality in asymptomatic healthy donors.

• Journal of Embryo Transfer
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• v.16 no.1
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• pp.53-60
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• 2001

This study was conducted to evaluate whether \"Royal jelly\" (RJ) added to Tris-buffer dilute contributed to supporting post-thaw viability and longevity of frozen canine spermatozoa. Two Japanese spitzs (2 to 4 years of age) were used as a semen donor. Semen was collected by manual masturbation and separated into 3 fractions. Only the sperm-rich fraction having sperm motility of more than 70%, containing sperm concentration of 2~4$\times$10$^{8}$ cells/ml and having dead or abnormal spermatozoa of less than 15% was used for the experiment. Each ejaculated semen was centrifuged at 400 $\times$ g for 5 min and then diluted in a Tris-buffer supplemented with 20 ml egg yolk (Ext I), 4% glycero1 and 1% Equex STM Paste (Ext II) or g1ycero1, Equex STM paste and RJ of various concentrations (Ext II-RJ). After freezing and thawing, viability of spermatozoa in Ext II -RJ containing 1% RJ immediately after thawing (67.5$\pm$9.6) was significantly lower than that of Ext II , Ext II -RJ containing 0.01 or 0.1% RJ (77.5$\pm$12.5, 78.7$\pm$8.2 and 80.0$\pm$6.3). However, Ext II-RJ containing 0.1% RJ yielded higher viability than Ext II, Ext II-RJ containing 0.01% at or 1% 1 h after thawing (69.5$\pm$8.1 vs. 55.0$\pm$12.9, 57.5$\pm$9.6 and 41.5$\pm$12.6; P<0.05). At 1 h after thawing, the viability of spermatozoa thawed in 7$0^{\circ}C$ (68.8$\pm$12.5) was significantly higher than that of spermatozoa thawed in 38$^{\circ}C$ (48.8$\pm$16.3), although there was no difference in the viability between both groups immediately after thawing (77.5$\pm$9.6 and 81.3$\pm$8.1). Post-thaw viability and longevity of post-thaw spermatozoa in Ext II-RJ containing 0.1% RJ was higher in those in Ext II at 1 h (65.0$\pm$12.9 vs. 42.5$\pm$12.6), 2 h (52.5$\pm$12.6 vs. 27.5$\pm$17.1) and 3 h (40.0$\pm$14.1 vs. 20.0$\pm$12.1) after thawing. These results indicated that addition of 0.1% af to Tris-buffer enhanced post-thaw viability and longevity of canine spermatozoa and this additive can be used for increasing the possibility of collision between spermatozoa and ova during insemination.emination.

• Korean Journal of Animal Reproduction
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• v.14 no.4
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• pp.237-244
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• 1990

These studies were carried out to investigate the effects of the follicles size, hormone supplementation, semen types and capacitation methods on in vitro maturation and fertilization of bovine follicular oocytes. The ovaries were obtained from slaughtered Korean Native cows. The follicular oocytes surrounded with cumulus cells were recovered by aspirating follicular fluid from the visible follicles of diameter 3~5mm. The follicular oocytes were cultured in TCM-199 medium containing hormones, FCS, ECS, BFF and MCC for 24~48hrs. in a incubator with 5% CO2 in air at 38.5$^{\circ}C$ and then matured oocytes were again cultured for 18~20hrs. with motile capacitated sperm in the TCF(Tyroide calcium-free) solution containing 200$\mu\textrm{g}$/ml of heparin. The results obtained in these experiments were summarized as follows : 1. The oocytes classified as "A, B, C, D and Degenerative" depending morphological integrity and those were 61.4%, 12.1%, 19.2%, 4.2% and 3.0% of the total oocytes recovered, respectively. The maturation and fertilization rate of the A, B, C class follicular oocytes, cultured in the TCM-199 medium supplemented with 10% FCS were 89.1%, 78.0%, 52.6% and 78.1%, 66.1%, 33.3%, respectively. 2. The average number of the follicular oocytes recovered from follicles size, 1~2mm, 3~5mm and above 5mm in dimeter were 67, 98 and 63, respectively. The maturation and fertilization rate of the follicular oocytes, cultured in the TCM-199 medium were 56.7%, 82.5%, 46.0% and 44.8%, 71.4%, 28.6%, respectively. 3. The maturation and fertilization rate of follicular oocytes, cultured in the TCM-199 medium supplemented with 5%, 10%, 15%, 20% FCS and hCG, HCG, $\beta$-estradiol were 76.0%~82.3% and 26.2%~70.0%, and those values were higher the supplementation of the hormone than the non-supplementation. 4. The fertilization and cleavage rate of the follicular oocytes, inseminated with spermatozoas of epididymis cauda, neat and frozen semen were 63.3%, 73.3%, 70.0% and 32.7%, 37.8%, 38.3, respectively. 5. The fertilization and cleavage rate of follicular oocytes, fertilized with capacitated spermatozoas by heparin, BFF and HIS methods were 70.0%, 53.8%, 34.2% and 38.3%, 23.1%, 17.1%, respectively. And the fertilization and cleavage rate were higher method of heparin than other methods.r methods.