• Korean Journal of Animal Reproduction
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• v.24 no.2
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• pp.209-216
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• 2000

This study was carried out to investigate the effects of breed, age of boar, season, parity and mating system on boar semen characteristics and fertilizing capacity. A total of 4181 sows and 199 boars of Durocs (D), Landraces (L), and Yorkshires (Y) were used for this experiment at Darby Artificicial Insemination Center from 1996 through 1999. Semen volume per ejaculate was largest in Landrace (266.8 $m\ell$), followed by Yorkshire, and was smallest in Duroc. Sperm motility did not show significant differences among the above breeds. Sperm concentration was lowest in Landrace (4.7$\times$10$^{9}$ sperm/$m\ell$) and was highest in Duroc (5.7$\times$10$^{9}$ sperm/$m\ell$). Semen volume per ejaculate according to the age of boars was largest at the age of 2 years, followed by the age of 4 and 3 years, and was smallest at the age of I year. Semen volume per ejaculate according to the season in boars was largest in winter (228.6 $m\ell$), followed by autumn and summer, and was smallest in spring. Sperm concentration was highest in spring (5.9$\times$10$^{9}$ sperm/$m\ell$), followed by summer and winter, and was lowest in autumn. The average litter weight at birth did not show any differences according to the mating type. But the number of pigs born alive per litter was largest (9.5 pigs) in the natural mating ＋ artificial insemination group, followed by the artificial insemination group (9.2 pigs), and was smallest (8.9 pigs) in the natural mating group (P＜0.01). The average litter weight at birth and number of pigs born alive per litter did not show any differences between the natural mating and artificial insemination. The L (♀)$\times$Y (♂) and L (♀)$\times$L (♂) matings show $\varepsilon$ d higher average litter weight at birth and number of pigs born alive per litter than the Y (♀) $\times$ Y (♂) and Y (♀) $\times$ L (♂) matings. The pigs in the 2~6th parities had higher average litter weight at birth and number of pigs born alive per litter than those in the 1 st and 7~9th parities.

• Korean Journal of Environmental Biology
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• v.38 no.2
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• pp.323-331
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• 2020

The method of natural spawning is very passive and inconvenient for the study of developmental engineering in marine medaka, Oryzias dancena. The optimum concentration of human chorionic gonadotropin (HCG) and carp pituitary extract (CPE) for ovulation and spawning, and the injection time for the artificial spawning of marine medaka were analyzed in this study. The success rate, survival rate, and hatching rate were highest with 100IU HCG kg^-1 BW and 5mg CPE L^-1 in both male and female marine medaka (p<0.05). After obtaining unfertilized eggs and sperm by the injection of HCG and CPE into the broodstock of marine medaka, artificial fertilization could be successfully achieved any time fertilized eggs are needed in this species. This result should be useful for developing a study program for marine medaka as an experimental animal.

• Journal of Aquaculture
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• v.9 no.2
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• pp.133-140
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• 1996

Hybird and allotriploid between female rainbow trout (Oncorhynchus mykiss) and male coho salmon (O. kisutch) were produced by artificial fertilization and heat shocks. Hatching and survival rate of allotriploid at 2 month after hatching was $77.6\%$ and $54.5\%$ respectively, and these rates clearly exceeded those of their hybrid. Cell and nuclear sizes of the erythrocyte of hybrid were intermidiate of their parents and those of allotriploid were larger than thier hybird. The somatic chromosome number of viable hybrid was 2n = 60 and that of allotriploid was $90\~93$ with chromosomal polymorphism. Allotriploid karyotpe was constituted by two sets of rainbow trout chromosome and one set of coho salmon chromosome.

• Journal of Embryo Transfer
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• v.18 no.2
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• pp.143-149
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• 2003

This study was carried out to cryopreserve and to investigate characteristics of semen in Hanwoo. Semen was obtained from bulls selected by Daekwanryeong Branch station. Semen was collected each morning of the experiment, placed in water jacketed tubes at 37$^{\circ}C$, and trans-ported to the research laboratory within 10 minutes. Semen was extended with Egg yolk-glycerol extender to contain 50${\times}$10$^{6}$ sperm/ml. Semen was cooled over a 6h period in water jacketed tubes from about 25 to 5$^{\circ}C$, Egg yolk-glycerol extender was added in one step at 5$^{\circ}C$. Semen was aspirated into 0.5ml straws, which were sealed with powder. Egg yolk-glycerol extender, which is used in Hanwoo sperm frozen and stored, semen from 13 Hanwoo bulls collected, the postthawed percentages of motile sperm were 65.7%. In semen characteristics of Hanwoo bulls, number of bulls volume are 5.7 ml and total cell count are 975${\times}$10$^{6}$ m1 ejaculate.

• Proceedings of the Korean Aquaculture Society Conference
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• 2003.10a
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• pp.64-64
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• 2003

대구 인공종묘 생산 기술 개발의 일환으로 수정란에 기생하여 폐사를 유발하는 세균 및 세균 감염 경로를 파악함으로써 수정란의 생존율과 부화율 향상을 위하여 본 실험을 실시하였다. 세균 감염 경로를 파악하기 위하여 난소와 정소 자체 세균 감염 여부를 조사하였다. 일반배지 (BHIA)와 비브리오 선택배지 (TCBS)를 사용하여 세균검사를 실시하였다. 난소의 세균 검사는 생식소 내부를 일부 절개한 후 멸균 roop를 찔러 세균을 검사하였고, 정소는 채정하기 전 복부를 절개하여 멸균 roop로서 적출하여 배지에 도말한 후 배양하였다. 수정란 내 세균 수는 수정란을 각각 20개씩 샘플하여 난 외부에 기생하는 세균을 제거하기 위하여 난소독제로 이용되고 있는 benzalkonium 0.1%로 1분간 소독한 후 멸균 생리식염수로 3회 세척하여 호모게나이즈하여 검사하였다. 호모게나이즈한 액 중 100${\mu}\ell$를 pipetting하여 일반배지(BHIA)에 도말하여 인큐베이터에서 배양하였다. 난소와 정소 내에서 세균이 검출됨에 따라 수정 시 세균 감염을 억제하기 위하여 자외선 살균해수와 일반해수를 수정액으로 사용하여 발생율을 비교 시험하였다. 수정 시간은 1분으로 동일하게 적용하였으며, 수정 용기는 멸균 처리된 일회용 100$m\ell$ 플라스틱 용기를 사용하였다. 수정 후 과다한 정자를 제거하기 위한 세란은 1$\ell$ 멸균 비이커에서 5회 30분 가량 실시하여 정자를 제거하였고 멸균 봉으로 저어주면서 수정란의 점착력을 제거하였다. 수정란은 100$\mu\textrm{m}$ 그물망으로 수정란 유실을 방지한 플라스틱 용기에 수용하여 유효수량 270$\ell$ FRP 수조에 수용하여 3$\ell$/min 환수하였고, 수온은 자연수온 1$^{\circ}C$로 유지하였다. 발생율은 만능투영기로 3회 측정하였다. 수정 후 세균과 기생충에 의한 수정란의 폐사를 억제하기 위하여 수정 직후, 수정 후 1일, 2일째 oxytetracycline과 iodine 처리에 따른 발생율 변화를 조사하였다. 발생율은 만능투영기로 조사하였고, 시험구별로 3회 측정하였다. 경과 일수별로 약제 처리는 약제 미처리 수정란 중 정상적인 발생이 이루어지고 있는 것을 선별하여 조사하였다. 약제 처리에 따른 배체 발생 단계는 수정 후 1일째는 상실기, 2일째는 포배기였다. 수정란 및 대구 자어에서 분리된 V. splendidus 에 의한 폐사를 예방하기 위해 in vivo에서 oxytetracycline 외 5종의 항생제를 대상으로 96well plate에서 최고 농도 250ppm부터 2 fold로 단계 희석하여 $25^{\circ}C$에서 48시간 배양하여 MIC를 조사하였다. 세균 감염경로 파악을 위하여 난소, 정소 및 정자에서 세균을 분리한 결과 일반배지 및 비브리오 선택배지에서 모두 균이 검출되었고, 균 동정 결과 터봇 자어에서 검출된 것으로 보고된(Gatesoupe et al., 1999) V. splendidus로 나타났다. 수정액과 정자 및 미수정란의 세균 분리 결과 일반배지에서 3$\times$10/ml ~ 7$\times$10/ml로 균이 검출되었다. 수정액을 일반해수와 자외선 살균 해수를 사용하여 발생율을 비교한 결과 수정 후 3일째 발생율은 자외선 살균해수 72.3%, 일반해수 52.7%였으며, 수정 후 7일째 40.9%와 25.1%로 자외선 살균해수가 유의적으로 높게 나타났다. 수정 후 경과 일수별로 oxytetracycline과 Iodine을 처리한 결과 수정 직후 처리한 시험구는 7일째 19.8%와 18.9%로 대조구 23.1%와 유의적인 차이가 없는 것으로 나타났다. 수정 후 1일째 처리한 시험구는 54.5%와 56.8%, 수정후 2일째 처리구는 47.9%와 50.6%로 두 시험구 모두 대조구와 수정 직후 처리구보다 유의적으로 높게 나타났다. 수정란 및 대구 자어에서 분리된 V. splendidus 에 의한 폐사를 예방하기 위해 in vivo에서 항생제 종류별 MIC 조사 결과 oxytetracycline이 0.48ppm으로 가장 효과가 높은 것으로 나타났다.

• 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.

• Journal of Embryo Transfer
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• v.16 no.1
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• pp.35-40
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• 2001

Artificial insemination (AI) with frozen or cooled semen is widely used in commercial fields of cattle and pig. Little is known about characteristics of canine sperm after freezing or cooling. For both practical and commercial goal, the canine semen treated with cooling and freezing should be carried out to exam the fundamentals, including sperm motility, survivability and fertilizing capacity. The aim of this study, thus, was to identify the effects of extended exposure to 4$0^{\circ}C$ on canine semen by motility, survivability, acrosomal changes following different duration. Fifteen ejaculates collected by digital manipulation twice per week from 3 dogs (Shih-Tzu) were divided to 16 aliquots after adding Tris-egg yolk (TE) buffer formulated by our laboratory, and cooled from 37 to 4$^{\circ}C$, by ramp rate of 0.6$^{\circ}C$/min. Each sample was evaluated by their motility, survivability and the acrosomal status at 0h (control), 2h, 12h and 1 d~10 d, respectively. The motility of spermatozoa was graded to 6 levels using the modified method of Seager. The survivability of sperm was assessed using an epifluorescence microscope after Fert/Light (Mole-cular Probes Inc.) staining. To estimate the proportion of the spermatozoa of intact acrosome, 200 spermatozoa were assessed in randomly selected fields, using epifluorescence microscope after FITC/PSA (Sigma) staining. At 2 h after cooling, the motility of most spermatozoa were assessed to be grade 0 and 1. At 12 h, high number of sperm were in grade 0 to 1, however, it was significantly (P<0.05) lower than that of 2 h. From 1 d to 4 d, ~50% of sperm was assessed to grade 0 to 1. On day 7, a little sperm were in grade 0 to 1. No sperm showed motility on day 10. Sperm motility was rapidly reduced by the percent of 10% of grade 0 to 1. From 2 h to 6 h, the number of live sperm was 90% and the sperm chilled for 10 days lived>50%. Acrosomal intact of spermatozoa exposed to 4$^{\circ}C$ for 2 h was 51%, supposed the sperm of control was 100%. Our results suggest that 1) this is easy to transfer and preservation for short periods 2) AI can be used by semen chilled for 6-Day.

• 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.

• Journal of Embryo Transfer
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• v.21 no.3
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• pp.255-262
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• 2006

The purpose of this study is to investigate the effects of vitrification in open pulled straws (OPS) methods on in vitro survival ability of porcine embryos. For in vitro maturation of immature oocytes, the porcine ovaries were collected from local slaughter-house. The cumulus-oocytes complexes were aspirated from 2 to 6 mm follicles. The collected oocytes were cultured for in vitro maturation in NCSU-23 medium with 5 mM hypotaurine, 0.57 mM cysteine, 10% porcine follicle fluid, 10 IU/ml PMSG and 10 IU/ml hCG for $21{\sim}22$ hrs. Then, the oocytes were more cultured $21{\sim}22$ hrs in vitro maturation in medium removed hormones. The frozen-thawed spermatozoa were washed by centrifugation 2 times for 10 min at 1,500 rpm in D-PBS with 5.56 mM glucose, 0.33 mM Na-pyruvate, 100 IU/ml penicillin, $100 {\mu}g/ml$ streptomycin and 4 mg/ml BSA. The fertilization medium used mTBM with 2 mM caffeine and 2 mg/ml BSA and adjusted to a pH of 7.2 to 7.4. The final concentration of spermatozoa was adjusted to $2.5{\times}10^6$cells/ml motile sperm during fertilization in vitro. At 8 hrs after insemination, the oocytes were transferred into NCSU-23 medium with 5.0 mM hypotaurine, 4 mg/ml BSA and 10 ng/ml EGF and cultured for 7 days. When the blastocysts of different stages were frozen-thawed by OPS methods, the proportions of embryos with normal morphology were significantly (p<0.05) higher in embryos frozen-thawed at expanded blastocyst stage (38.9%) than in early blastocyst stage (28.3%). On the other hand, the proportions of embryos damaged after frozen-thawing were significantly (p<0.05) higher in embryos frozen at early blastocyst stages than in expanded blastocyst stage. In another experiment, the normal embryos morphology after frozen- thawing were further cultured for 48 hrs. After culture, the proportions of embryos hatched were 6.7, 20.0 and 33.3% for embryos frozen-thawed at early blastocyst, mid-blastocyst and expanded blastocyst stages. These finding indicate the possible broader application for OPS methods, as frozen-thawed embryos may be accompanied by developmental stage according to requirements of the survival ability after freezing of blastocyst stage in the pig.

• Korean Journal of Animal Reproduction
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• v.10 no.1
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• pp.19-26
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• 1986

This experiment was carried out to investigate the effect of bovine serum ablumin (BSA), sugars, glycerol equilibration time, straw size and thawing method on the survival index and the morphology of frozen boar spermatozoa. The results obtained were summarized as follow: 1. When the semen frozen in BF5 dilutor as pellet form was thawed in BTS at 37$^{\circ}$and 50$^{\circ}C$, BF5 dilutor with fructose showed higher sperm survival index than that with dextrose, however, when the semen was thawed on dry test tube at 37$^{\circ}C$, BF5 dilutor with sucrose showed higher sperm survival index than with other sugars. 2 When the semen forzen in BF5 dilutor with straw and thawed at 37$^{\circ}C$, BF5 dilutor with dextrose showed higher sperm survival index than those with other sugars, and there was no difference in sperm survival index between 0.5 and 1.0 ml straws. 3. The sperm survival index of frozen sperm was significantly (P<0.05) improved due to addition of BSA (0.05%) to BF5 dilutor. 4. When the extended semen with BF5 dilutor contatining 0.01 to 0.05% of BSA was frozen in the straw, the semen without glycerol equilibration showed significantly (P<0.05) higher sperm survival index than those with 2, 4 and 6 hrs glycerol equilibration time. 5. The sperm frozen in BF5 dilutor with dextrose or fructose, sucrose and raffinose showed 77 to 88% in normal acrosome rate and no difference among sugars. 6. The frozen semen showed lower normal acrosome rate than the first and second diluted semen, whereas the frozen semen showed higher swollen, damaged and missing acrosome rate than the first and second diluted semen. 7. Damaged and missing acrosome rate of sperm head due to freezing was somewhat inhibited by addition of BSA (0.01 to 0.05) to the BF5 dilutor.